Maytansinoid derivatives

ABSTRACT

Disclosed herein are maytansinoid drug linker derivatives which can be linked to a antigen binding unit (Abu), and maytansinoid drugs linked with an antigen binding unit (Drug-Linker-Antigen binding Unit: D-L-Abu), for targeted delivery to disease tissues. D-L-Abu, D-L-Abu derivatives, and methods relating to the use of such drug conjugates to treat antigen positive cells in cancers and immunological disorders are provided.

The present invention provides maytansinoid derivatives, includingmaytansinoid derivatives for linking to antigen binding unit andmaytansinoid drug linked with antigen binding unit, for targeteddelivery to disease cells. Also provided are related compositions andmethods for treating diseases, such as cancers and immunologicaldisorders.

BACKGROUND

Maytansinoids are highly cytotoxic compounds which inhibit the formationof microtubule protein polymerization (Remillard, et al., Science 189,1002-1005 (1975)). Maytansine was first isolated by Kupchan et al. (J.Am. Chem. Sci 94:1354-1356 (1972)) from the east African shrub Maytenusserrata. Maytansinoids including maytansinol and C-3 esters ofmaytansinol were also produced by certain microbes (U.S. Pat. No.4,151,042). Various analogues of maytansinol with different cytotoxicityhave also been prepared by synthetic chemistry (for review see Chem.Pharm. Bull. 52(1) 1-26 (2004)). Examples of mytansinoids includemaytansine, mertansine (DM1), DM3 and DM4. Maytansine is a strongmitotic inhibitor and shows significant inhibitory activity againstmultiple tumors including Lewis lung carcinoma and B-16 melanocarcinomasolid murine tumor models. Maytansine was reported to inhibit the humanacute lymphoblastic leukemia line C.E.M. at concentrations as low as10⁻⁷ μg/mL (Wolpert-DeFillippes et al., Biochem. Pharmacol. 1735-1738(1975)). It also showed to be 100- to 1000-fold more cytotoxic thanconventional chemotherapeutic agents like methotrexate, daunorubicin,and vincristine (U.S. Pat. No. 3,896,111).

Ansamitocins, the bacterial maytansinoids, show an activity spectrum andeffective dosage range similar to maytansine. They inhibit P388 leukemiaat daily doses as low as 0.8 μg/kg. Ansamitocin P3 (AP3) was also shownto be effective against multiple cancer cell lines (for review seeAlkaloids, vol. 2, 149-204 (1984); Chem. Pharm. Bull. 52(1) 1-26(2004)). The maytansinol C-3 esters with N-methyl-L-alanine derivativesare found to be much more cytotoxic than the corresponding esters ofsimple carboxylic acid and to be 100 times more cytotoxic than theirepimers corresponding to N-methyl-D-alanine (U.S. Pat. Nos. 4,137,230;4,260,608; Kawai, et al., Chem. Pharm. Bull. 32: 3441-3451 (1984);Widdison, et al., J. Med. Chem. 49: 4392-4408 (2006)).

Maytansinoids were expected to have the capacity to treat many differentcancers due to their highly toxic nature and the in vitro activitiesagainst multiple cancer cell lines. However, the toxicity also made thisclass of compounds not favorable in human clinical trials as the sideeffects were intolerable for many patients (Issel et al., Cancer Treat.Rev. 199-207 (1978)). Accordingly, targeted delivery of cytotoxiccompounds to cancer cells by conjugating toxic drugs to monoclonalantibodies (ADC for antibody drug conjugate) is proposed in order toreduce the side effects. Certain conjugates of cytotoxic drugs such asmaytansinoids, auristatins, anthracyclins, duocarmycins, etc. withantibodies are being evaluated in preclinical or clinical studies in thetreatment of diseases.

Antibody drug conjugates (ADCs) are composed of three key elements:antibody, linker, and drug. The selection of a particular antibody anddrug will have a great impact on the efficacy and safety depending onthe particular disease. Linker stability and the method by which thedrug is conjugated to the antibody plays a critical role in the successor failure of the ADC drug development.

The efficacy of an ADC depends in part on combination of a variety ofparameters, involving not only the specificity of the antibody and thepotency of drugs, but also the linker's stability or sensitivity tocleavage, the cell surface triggered the internalization, trafficking,and subsequent release of the active cytotoxic payload. Thus, ADCcomprising different drug linkers or with different antibodies againstthe same target can vary significantly in their utility.

SUMMARY OF THE INVENTION

The present invention provides maytansinoid drug derivatives which canbe linked to a antigen binding unit (Abu), and maytansinoid drug linkedwith antigen binding unit (Drug-Linker-Antigen binding Unit: D-L-Abu),for targeted delivery to disease cells or tissues. D-L-Abu, D-L-Abuderivatives, and methods relating to the use of such drug conjugates totreat antigen positive cells in cancers and immunological disorders arealso provided. The antigen binding unit, Abu, such as an antibody, orother targeting moiety in the D-L-Abu, binds to an antigen in thedisease cells or tissues. A drug conjugated to the Abu exerts acytotoxic, cytostatic, or immunosuppressive effect on theantigen-expressing cells to treat or prevent recurrence ofantigen-positive cancers or immunological disorders. The presenttechnology provides drug-linker-antigen binding unit exerting cellularinhibitory or killing effect on the antigen positive cells, whileminimizing the undesirable side effects, such as bystander killingeffects on antigen negative cells.

In one aspect, provided are maytansinoid derivative compounds capable ofconjugation to an antigen binding unit via a linker that is not acidlabile, not peptidase cathepsin sensitive, and does not contain adisulfide bond. Such derivatives may also be referred to as“drug-linkers.” In some embodiments, the linker modified maytansinoidcompounds is a maytansinoid N₂′-deacetyl-N₂′-(6-maleimido-1-oxo-hexyl)maytansine, or its derivatives, wherein the linker is not acid labile,not peptidase cathepsin sensitive, and does not contain a disulfidebond. Such linkers are contemplated to provide stability to the D-L-Abuprior to endocytosis, such as during circulation, to prevent prematuredegradation of D-L-Abu and release of the toxic drug, thus minimizes thetoxic effect of the drug.

In some embodiments, provided herein is a maytansinoid derivative ofFormula I or I-1, formula II or II-1:

or a salt thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   each Z is independently hydrogen or C₁-C₄ alkyl, or the two Z        with the carbon atom to which they are attached form a C═O; and    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo.

In another aspect, provided is a composition comprising theabove-described maytansinoid compound and an antigen binding unitcapable of being conjugated with each other.

In another aspect, provided is an antigen binding unit conjugated with amaytansinoid compound, wherein the maytansinoid compound is modified,and linked to an antigen binding unit via a linker that is not acidlabile, not peptidase cathepsin sensitive, and does not contain adisulfide bond.

In some embodiments, provided herein is a maytansinoid linker antigenbinding unit conjugate of Formula Ia, Ia-1, IIa or IIa-1:

or a pharmaceutically acceptable salt or solvate thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;    -   each Z is independently hydrogen or C₁-C₄ alkyl, or the two Z        with the carbon atom to which they are attached form a C═O;    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR—, —C(O)—, —C(═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; Preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo; and    -   Abu is an antigen binding unit.

In some embodiments, provided are compounds of the formula:

or a pharmaceutically acceptable salt or solvate thereof.

In another aspect, provided is a composition comprising theabove-described maytansinoid linker antigen binding unit conjugate.

In another aspect, provided is a method of preparing the above-describedmaytansinoid linker antigen binding unit conjugate which methodcomprises contacting an antigen binding unit with one or moremaytansinoid compounds described herein capable of being conjugated tothe antigen binding unit.

In another aspect, disclosed herein is a compound of Formula Ib, Ib-1,IIb or IIb-1:

or a salt thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   each Z is independently hydrogen or C₁-C₄ alkyl, or the two Z        with the carbon atom to which they are attached form a C═O;    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo; and    -   AA is an amino acid, such as a cysteine or a thiolated amino        acid, such as a thiolated lysine.

In some embodiments, provided are compounds of the formula:

In another aspect, provided is a composition comprising theabove-described maytansinoid compound of Formula Ib, Ib-1, IIb or IIb-1.

In another aspect, provided is a method for targeting a maytansinoid toantigen positive cells or tissues with an antigen binding unitconjugated with maytansinoid described herein.

In another aspect, provided is an antigen binding unit, Bat0206comprising an anti-EGFR light chain having an amino acid sequence of SEQID NO: 1 and an anti-EGFR heavy chain having an amino acid sequence ofSEQ ID NO: 2.

In another aspect, provided is a maytansinoid linker antigen bindingunit conjugate wherein the antigen binding unit is Bat0206.

In another aspect, provided is a method for treatment of proliferativedisorders such as tumors, inflammatory or immunologic diseases such asgraft rejections, and other diseases that can be treated by targetedtherapy in a subject in need of the treatment, wherein the disease ischaracterized by cells comprising an antigen that binds to an antigenbinding unit, said method comprising administering to the subject aneffective amount of the antigen binding unit conjugated with one or moremaytansinoid compound described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Sephadex G25(M) chromatograph of purification ofBatansine-0206.

FIG. 2 shows Sephadex G25(M) chromatograph of purification ofBatansine-1206.

FIG. 3 shows Phenyl Sepharose FF column separation of Batansine-0206.

FIG. 4 shows Sephadex G25(M) column separation of Batansine-0606.

FIG. 5 shows Phenyl Sepharose FF column separation of Batansine-0606.

FIG. 6 shows the effects of the metabolites of prodrug antibodymaytansinoid conjugates on the tubulin polymerization.

FIG. 7 shows that Bat0206 and Batansine-0206 inhibited tumor cellgrowth.

FIG. 8 shows that Bat0206, cetuximab, Batansine-0206 has no inhibitoryeffect on EGFR negative cells.

FIG. 9 shows the inhibitory effect of D-Lmcc-Bat0206 towards MDA-MB-468cells.

FIG. 10 shows that Bat0206, cetuximab, D-Lmcc-Bat0206 has no inhibitoryeffect on EGFR negative cells.

FIGS. 11 and 12 shows that D-Lmcc-Bat0206 eradicated A431 tumorxenographs.

FIG. 13 shows that D-Lmcc-Bat0206 and D-Lspp-Bat0206 inhibited A431tumor cells.

FIG. 14 shows D-Lmcc-Bat0206 and D-Lspp-Bat0206 inhibited A549 tumorcells.

FIGS. 15A and 15B show the amino acid sequences of anti-EGFR antibody.

FIGS. 16 and 17 show the processing of D-Lmcc-bat0606 and Batanine-0606using Sephadex G-25.

FIGS. 18 and 19 show the size exclusion chromatograph of D-Lmcc-bat0606and Batanine-0606 respectively.

FIG. 20 shows the inhibitory effects of Bat0606 and Batanine-0606 onHer2 (SK-BR-3) cells.

FIG. 21 shows that Bat0606 and Batanine-0606 did not inhibit A549.

FIG. 22 shows the inhibitory effect of D-Lmcc-Bat0606 on SK-BR-3.

FIG. 23 shows that Bat0606 and D-Lmcc-Bat0606 did not inhibit A549cells.

FIG. 24 shows the inhibitory effects of Bat1206 and Batansine-1206 onRaji cells.

FIG. 25 shows that Bat1206 and Batansine-1206 had no effects on A431cells.

FIG. 26 shows that D-Lmcc-Bat1206 inhibited Raji cell growth.

FIG. 27 shows the purification of Batansine-1206 on SEC-HPLC.

FIG. 28 shows the non-reduced SDS-PAGE of Bat1206 and D-Lmcc-Bat1206.

FIG. 29 shows Batansine-0606 eradicated human NCI-N87 tumor in mousexenografts.

FIG. 30 shows Batansine-0606 eradicated human BT474 tumor in mousexenografts.

FIG. 31 shows the ESI-MS spectra of the metabolite (Batansine-Cysteine)from the prodrug Batansine-0606.

FIG. 32 shows the ESI-MS spectra of the two diastereomeric metabolites(MDC-MCC-Lysine) from the prodrug D-Lmcc-Bat0606.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “acompound” includes a plurality of compounds.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% or plus or minus 5%, orplus or minus 1% of the particular term.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this invention.

As used herein, “maytansinoid” refers to a maytansine analogue,including stereoisomers thereof. Maytansine can be isolated from plantsof the genus Maytenus U.S. Pat. No. 3,896,111). It is of the formula:

Maytansinoids are compounds having the ring structure of maytansine withone or more modifications of the substituents on the ring.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.C_(v) alkyl wherein v is an integer represents an alkyl having vcarbons. This term includes, by way of example, linear and branchedhydrocarbyl groups such as methyl(CH₃—), ethyl(CH₃CH₂—),n-propyl(CH₃CH₂CH₂—), isopropyl((CH₃)₂CH—), n-butyl(CH₃CH₂CH₂CH₂—),isobutyl((CH₃)₂CHCH₂—), sec-butyl((CH₃)(CH₃CH₂)CH—), t-butyl((CH₃)₃C—),n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl((CH₃)₃CCH₂—). “Alkylene” is adivalent saturated aliphatic hydrocarbyl groups having from 1 to 10carbon atoms and preferably 1 to 6 carbon atoms.

“Alkenyl” refers to straight or branched hydrocarbyl groups having from2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having atleast 1 and preferably from 1 to 2 sites of vinyl(>C═C<) unsaturation.Such groups are exemplified, for example, by vinyl, allyl, andbut-3-en-1-yl. Included within this term are the cis and trans isomersor mixtures of these isomers.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of acetylenic (—C≡C—)unsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

“Amino” refers to the group —NR′R″ where R′ and R″ are independentlyselected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, and wherein R′ and R″ areoptionally joined, together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group, provided that R′ and R″are both not hydrogen, and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. When R′ is hydrogen andR″ is alkyl, the substituted amino group is sometimes referred to hereinas alkylamino. When R′ and R″ are alkyl, the substituted amino group issometimes referred to herein as dialkylamino. When referring to amonosubstituted amino, it is meant that either R′ and R″ is hydrogen butnot both. When referring to a disubstituted amino, it is meant thatneither R′ and R″ are hydrogen.

“Amino acid” refers any compound, whether natural, unnatural orsynthetic, which comprises both an amino group and a carboxy group.Examples of amino acid include, but are not limited to glycine(NH₂CH₂COOH), cysteine, alanine, N-methyl-L-alanine, including both theD and L optical isomers. “Amino acid side chain” refers to thesubstituent that replaces a hydrogen of the methylene group of glycineor glycine derivatives, such as N-alkylglycine or glycine esters.Examples of an amino acid side chain include, but are not limited to theside chains of the natural amino acids, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, heteroaryl, substituted heteroaryl,heterocyclic, and substituted heterocyclic.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryl groupsinclude phenyl and naphthyl.

“Carbonyl” refers to the divalent group —C(O)— which is equivalent to—C(C═O)—.

“Carboxy” or “carboxyl” refers to —COOH or CO₂H or salts thereof.

“Carboxylic acid” refers to a compound having at least one carboxy.

“Cyano” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. One or more of the rings can be aryl, heteroaryl, orheterocyclic provided that the point of attachment is through thenon-aromatic, non-heterocyclic ring carbocyclic ring. Examples ofsuitable cycloalkyl groups include, for instance, adamantyl,cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl. Other examples ofcycloalkyl groups include bicycle[2,2,2,]octanyl, norbornyl, andspirobicyclo groups such as spiro[4.5]dec-8-yl:

Cycloalkylene refers to a cyclic alkylene.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple cyclic rings and having atleast one >C═C< ring unsaturation and preferably from 1 to 2 sitesof >C═C< ring unsaturation.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo andpreferably is fluoro or chloro.

“Haloalkyl” refers to alkyl groups substituted with 1 to 5, 1 to 3, or 1to 2 halo groups, wherein alkyl and halo are as defined herein.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring.

Such heteroaryl groups can have a single ring (e.g., pyridinyl or furyl)or multiple condensed rings (e.g., indolizinyl or benzothienyl) whereinthe condensed rings may or may not be aromatic and/or contain aheteroatom provided that the point of attachment is through an atom ofthe aromatic heteroaryl group. In one embodiment, the nitrogen and/orthe sulfur ring atom(s) of the heteroaryl group are optionally oxidizedto provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties.Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl, thiophenyl,and furanyl.

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or partially saturated, but not aromatic, grouphaving from 1 to 10 ring carbon atoms and from 1 to 4 ring heteroatomsselected from the group consisting of nitrogen, sulfur, or oxygen.Heterocycle encompasses single ring or multiple condensed rings,including fused bridged and spiro ring systems. In fused ring systems,one or more the rings can be cycloalkyl, aryl, or heteroaryl providedthat the point of attachment is through the non-aromatic ring. In oneembodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic groupare optionally oxidized to provide for the N-oxide, sulfinyl, orsulfonyl moieties.

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, dihydroindole, indazole,purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,and tetrahydrofuranyl.

“Substituted alkyl,” “substituted alkenyl,” “substituted alkynyl,”“substituted cycloalkyl,” “substituted cycloalkenyl,” “substitutedaryl,” “substituted heteroaryl” or “substituted heterocyclic” refers toalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl orheterocyclic groups, respectively, which are substituted with 1 to 5,preferably 1 to 3, or more preferably 1 to 2 substituents selected fromthe group consisting of alkyl, halo alkyl, —O—R²⁰, —S—R²⁰, alkenyl,alkynyl, —C(═O)R²⁰, —C(═S)R²⁰, —C(═O)OR²⁰, —NR²⁰C(═O)R²¹, —OC(═O)R²¹,—NR²⁰R²⁰, —C(═O)NR²⁰R²⁰, —C(═S)NR²⁰R²⁰, —NR²⁰R²⁰C(═O)NR²⁰R²⁰,—NR²⁰C(═S)NR²⁰R²⁰, —OC(═O)NR²⁰R²⁰, —SO₂NR²⁰R²⁰, —OSO₂NR²⁰R²⁰,—NR²⁰SO₂NR²⁰R²⁰, —C(═N R²⁰)NR²⁰R²⁰, aryl, —NR²⁰C(═O)OR²¹, —OC(═O)OR²¹,cyano, cycloalkyl, cycloalkenyl, —NR²⁰C(═NR²⁰)NR²⁰R²⁰, halo, hydroxy,heteroaryl, heterocyclic, nitro, —SO₃H, —SO₂R²¹, and —OSO₂R²¹, whereineach R²⁰ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic or two R²⁰ with the atom(s) bound thereto form aheterocyclic ring, and R²¹ is selected from the group consisting ofalkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O) or (—O⁻).

“Spiro ring systems” refers to bicyclic ring systems that have a singlering carbon atom common to both rings.

“Thiol” refers to the group —SH.

“Thiocarbonyl” refers to the divalent group —C(S)— which is equivalentto —C(═S)—.

“Thione” refers to the atom (═S).

“Compound” or “compounds” as used herein is meant to include thestereoiosmers and tautomers of the indicated formulas.

“Stereoisomer” or “stereoisomers” refer to compounds that differ in thechirality of one or more stereocenters. Stereoisomers includeenantiomers and diastereomers.

“Tautomer” refer to alternate forms of a compound that differ in theposition of a proton, such as enol-keto and imine-enamine tautomers, orthe tautomeric forms of heteroaryl groups containing a ring atomattached to both a ring —NH— moiety and a ring ═N— moiety such aspyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.

“Solvate” refer to an association of a solvent with a compound, in thecrystalline form. The solvent association is typically due to use of thesolvent in the synthesis, crystallization, and/or recrystallization ofthe compound. “Solvate” includes hydrate which is an association ofwater with a compound, in the crystalline form.

“Patient” or “subject” refers to mammals and includes humans andnon-human mammals.

“Pharmaceutically acceptable salt” refers to pharmaceutically acceptablesalts of a compound, which salts are derived from a variety of organicand inorganic counter ions well known in the art and include, by way ofexample only, when the molecule contains an acidic functionality, saltsof organic or inorganic bases, such as sodium, potassium, calcium,magnesium, ammonium, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and tetraalkylammonium,and the like; and when the molecule contains a basic functionality,salts of organic or inorganic acids, such as hydrochloride,hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. Othernon-limiting examples of acids include sulfuric acid, nitric acid,phosphoric acid, propionic acid, glycolic acid, pyruvic acid, malonicacid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, p-toluenesulfonic acid, salicyclic acid and the like.

“Treating” or “treatment” of a disease in a patient refers to (1)preventing the disease from occurring in a patient that is predisposedor does not yet display symptoms of the disease; (2) inhibiting thedisease or arresting its development; or (3) ameliorating or causingregression of the disease.

“Effective amount” is intended to mean an amount of an active compoundor pharmaceutical agent that elicits the biological or medicinalresponse in a tissue, system, animal, individual or human that is beingsought by a researcher, veterinarian, medical doctor or other clinician,which includes treating a disease.

Drug Derivatives for Conjugation with an Antigen Binding Unit

In one aspect, disclosed herein is a maytansinoid derivative having alinking group capable of conjugating to an antigen binding unit (Abu),by forming a linker that is not acid labile, not peptidase cathepsinsensitive, and does not contain a disulfide bond.

Maytansinoids suitable for attaching the linking group includemaytansinol and maytansinol analogues and can be isolated from naturalsources according to known methods, produced using biotechnologies (seee.g., Yu et al., 99 PNAS 7968-7973 (2002)), or prepared syntheticallyaccording to known methods (see e.g., Cassady et al., Chem. Pharm. Bull.52(1) 1-26 (2004)).

Certain examples of suitable maytansinol analogues include:

-   -   (1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH        reduction of ansamytocin P2);    -   (2) C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat.        Nos. 4,361,650 and 4,307,016) (prepared by demethylation using        Streptomyces or Actinomyces or dechlorination using lithium        aluminium hydride (LAH));    -   (3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat.        No. 4,294,757) (prepared by acylation using acyl chlorides);    -   (4) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction        of maytansinol with H₂S or P₂S₅);    -   (5) C-14-hydroxymethyl(CH₂OH) or acyloxymethyl(CH₂OC(═O)phenyl        or CH₂OC(═O)(C₁-C₅ alkyl)) (U.S. Pat. No. 4,331,598) (prepared        from Nocardia);    -   (6) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by        the conversion of maytansinol by Streptomyces);    -   (7) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929)        (isolated from Trewia nudlflora);    -   (8) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348)        (prepared by the demethylation of maytansinol by Streptomyces);        and    -   (9) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the        titanium trichloride/LAH reduction of maytansinol).

Many positions on maytansinol can be useful as the linkage position,depending upon the type of linker. For example, for forming an esterlinkage, the C-3 position having a hydroxyl group, the C-14 positionmodified with hydroxymethyl, the C-15 position modified with a hydroxylgroup and the C-20 position having a hydroxyl group are all suitable. Insome embodiments, the linkage position is the C-3 position.

In some embodiments, provided herein is a maytansinoid derivative ofFormula I or I-1:

or a salt thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   each Z is independently hydrogen or C₁-C₄ alkyl, or the two Z        with the carbon atom to which they are attached form a C═O; and    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR—, —C(O)—, —C(═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo.

In some embodiments, the compound of Formula I is

In some embodiments, the compound of Formula I-1 is

In some embodiments, the compound of Formula I is the isomer:

In some embodiments, the compound of Formula I-1 is the isomer:

In some embodiments, provided herein is a maytansinoid derivative ofFormula II or II-1.

or a salt thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   each Z is independently hydrogen or C₁-C₄ alkyl, or the two Z        with the carbon atom to which they are attached form a C═O; and    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; Preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo.

In some embodiments, the compound of Formula II is

In some embodiments, the compound of Formula II-1 is

In some embodiments, the compound of Formula II is the isomer:

In some embodiments, the compound of Formula II-1 is the isomer:

In some embodiments, X is hydrogen. In some embodiments, X is chloro. Insome embodiments, Y is hydrogen. In some embodiments, R¹ is hydrogen. Insome embodiments, R² is methyl. In some embodiments, R³ is methyl. Insome embodiments, R⁴ is —OH. In some embodiments, R⁷ is an amino acidside chain. In some embodiments, R⁷ is methyl. In some embodiments, R⁸is methyl.

In some embodiments, L is unsubstituted C₁-C₂₀ alkylene. In someembodiments, L is substituted C₁-C₂₀ alkylene. In some embodiments, L isunsubstituted C₁-C₁₀ alkylene. In some embodiments, L is substitutedC₁-C₁₀ alkylene. In some embodiments, L is —(CH₂)₅—. In someembodiments, L is unsubstituted C₁-C₂₀ alkylene wherein one or two ofthe —CH₂— groups are independently replaced with C₃-C₈ cycloalkylene,—O—, —S—, —NR⁸—, —C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—. In someembodiments, L is substituted C₁-C₂₀ alkylene wherein one or two of the—CH₂— groups are independently replaced with C₃-C₈ cycloalkylene, —O—,—S—, —NR⁸—, —C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—. In someembodiments, when more than one —CH₂— groups are replaced, the —CH₂—groups are not adjacent to each other.

In some embodiments, provided is a compound of Formula III or III-1:

or a salt thereof,wherein

-   -   X is H or Cl;    -   Y is H or methyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁-C₆ alkyl; and    -   L is selected from C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, and        C₁-C₂₀ alkylene wherein one or more of the —CH₂— groups are        independently replaced with C₃-C₈ cycloalkylene, —O—, —S—, —NR—,        —C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—.

In some embodiments, the compound is of Formula IV or IV-1:

or a salt thereof,wherein

-   -   X is H or Cl;    -   Y is H or methyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁-C₆ alkyl; and    -   m is selected from an integer of 1 to 20, preferably 1 to 10,        more preferably 5 to 10.

In some embodiments, the compound isN₂-deacetyl-N₂-(6-maleimido-1-oxo-hexyl)maytansine, named batansine, ora salt thereof. Batansine is represented by Formula V:

In some embodiments, the compound of Formula I or I-1 is selected from:

or a salt thereof.

Drug Linker Antigen Binding Unit Conjugates

In another aspect, disclosed herein is a maytansinoid conjugated with anantigen binding unit (Abu) via a linker (drug linker Abu conjugate).

In exemplary embodiments, the drug linker Abu conjugate is amaytansinoid conjugated to an antibody via a linker that is neitheracid-labile, cathepsin sensitive, nor containing a disulfide bond.

In some embodiments, provided herein is a maytansinoid linker antigenbinding unit conjugate of Formula Ia or Ia-1:

or a pharmaceutically acceptable salt or solvate thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;    -   each Z is independently hydrogen or C₁-C₄ alkyl, or the two Z        with the carbon atom to which they are attached form a C═O;    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; Preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo; and    -   Abu is an antigen binding unit.

In some embodiments, the compound of Formula Ia or Ia-1 is

In some embodiments, the compound of Formula Ia is:

and the compound of Formula Ia-1 is:

In some embodiments, provided herein is a maytansinoid linker antigenbinding unit conjugate of Formula IIa or IIa-1:

or a pharmaceutically acceptable salt or solvate thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;    -   each Z is independently hydrogen or C₁-C₄ alkyl, or the two Z        with the carbon atom to which they are attached form a C═O;    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; Preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo; and    -   Abu is an antigen binding unit.

In some embodiments, the compound of Formula IIa or IIa-1 is

In some embodiments, the compound of Formula IIa is:

The compound of Formula IIa-1 is:

In some embodiments, X is hydrogen. In some embodiments, X is chloro. Insome embodiments, Y is hydrogen. In some embodiments, R¹ is hydrogen. Insome embodiments, R² is methyl. In some embodiments, R³ is methyl. Insome embodiments, R⁴ is —OH. In some embodiments, R⁷ is an amino acidside chain. In some embodiments, R⁷ is methyl. In some embodiments, R⁸is methyl.

In some embodiments, L is unsubstituted C₁-C₂₀ alkylene. In someembodiments, L is substituted C₁-C₂₀ alkylene. In some embodiments, L isunsubstituted C₁-C₁₀ alkylene. In some embodiments, L is substitutedC₁-C₁₀ alkylene. In some embodiments, L is —(CH₂)₅—. In someembodiments, L is unsubstituted C₁-C₂₀ alkylene wherein one or two ofthe —CH₂— groups are independently replaced with C₃-C₈ cycloalkylene,—O—, —S—, —NR⁸—, —C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—. In someembodiments, L is substituted C₁-C₂₀ alkylene wherein one or two of the—CH₂— groups are independently replaced with C₃-C₈ cycloalkylene, —O—,—S—, —NR⁸—, —C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—. In someembodiments, when more than one —CH₂— groups are replaced, the replaced—CH₂— groups are not adjacent to each other.

In some embodiments, provided is a compound of Formula IIIa or IIIa-1:

or a pharmaceutically acceptable salt or solvate thereof,wherein

-   -   X is H or Cl;    -   Y is H or methyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁-C₆ alkyl;    -   p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo; and    -   Abu is an antigen binding unit.

In some embodiments, the compound is of Formula IVa or IVa-1:

or a pharmaceutically acceptable salt or solvate thereof,wherein

-   -   X is H or Cl;    -   Y is H or methyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁-C₆ alkyl;    -   p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;    -   m is selected from an integer of 1 to 20, preferably 1 to 10,        more preferably 5 to 10; and    -   Abu is an antigen binding unit.

In some embodiments, the compound is of Formula Va:

or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the compound of Formula Ia or Ia-1 is selectedfrom:

In some embodiments, the compound of Formula Ia or Ia-1 is selectedfrom:

or a pharmaceutically acceptable salt thereof.

The maytansinoid component of the maytansinoid derivatives having alinking group capable of conjugating to an antigen binding unit (Abu) orthe maytansinoid linker antigen binding unit conjugates can besubstituted by other suitable cytotoxic agents, for example, anauristatin, a DNA minor groove binding agent, a DNA minor groovealkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, apuromycin, a dolastatin, and a vinca alkaloid. Other suitable cytotoxicagents include anti-tubulin agents, such as an auristatin, a vincaalkaloid, a podophyllotoxin, a taxane, a baccatin derivative, acryptophysin, a maytansinoid, a combretastatin, or a dolastatin. In someembodiments, the cytotoxic agent is AFP, MMAF, MMAE, AEB, AEVB,auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16,camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B,nocodazole, colchicines, colcimid, estramustine, cemadotin,discodermolide, maytansine, DM-1, DM-3, DM-4, or eleutherobin. Suitableimmunosuppressive agents include, for example, gancyclovir, etanercept,cyclosporine, tacrolimus, rapamycin, cyclophosphamide, azathioprine,mycophenolate mofetil, methotrexate, cortisol, aldosterone,dexamethasone, a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor,or a leukotriene receptor antagonist. In some embodiments, the cytotoxicagent is AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel,docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, dolastatin-10, echinomycin,combretatstatin, chalicheamicin, maytansine, DM-1, DM-3, DM-4, ornetropsin.

The maytansinoid component of the maytansinoid derivatives having alinking group capable of conjugating to an antigen binding unit (Abu)and the maytansinoid linker antigen binding unit conjugates can also besubstituted by a suitable immunosuppressive agent, for example,gancyclovir, etanercept, cyclosporine, tacrolimus, rapamycin,cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate,cortisol, aldosterone, dexamethasone, a cyclooxygenase inhibitor, a5-lipoxygenase inhibitor, or a leukotriene receptor antagonist.

Antigen Binding Units

The antigen binding units may be of any kind presently known, or thatbecome known, and include peptides and non-peptides. Generally, thesecan be antibodies (for example, monoclonal antibodies), lymphokines,hormones, growth factors, vitamins, nutrient-transport molecules (suchas transferrin), or any other cell-binding molecules or substances thatspecifically bind a target, derived from antibodies, antibody fragments,cell specific ligands, derived from peptides, carbohydrates, natural orsynthetic chemical compounds; or a pharmaceutically acceptable salt orsolvate thereof.

Antigen binding units include ragments of antibodies (polyclonal andmonoclonal) such as Fab, Fab′, F(ab′)₂, and Fv (see, e.g., Parham, J.Immunol. 131:2895-2902 (1983); Spring et al., J. Immunol. 113:470-478(1974); Nisonoff et al., Arch. Biochem. Biophys. 89:230-244 (1960));domain antibodies (dAbs) and antigen-binding fragments thereof,including camelid antibodies (see, e.g., Desmyter et al., Nature Struct.Biol, 3:752 (1996)); shark antibodies called new antigen receptors(IgNAR) (see, e.g., Greenberg et al., Nature, 374:168 (1995); Stanfieldet al. Science 305:1770-1773 (2004)).

Monoclonal antibody techniques allow for the production of antigenbinding unit in the form of specific monoclonal antibodies. Particularlywell known in the art are techniques for creating monoclonal antibodiesproduced by immunizing mice, rabbits, or any other mammal with theantigen of interest such as the tumor specific antigens isolated fromthe target cell. Another method of creating antigen binding unit isusing phage libraries of scFv (single chain variable region),specifically human scFv (see, e.g., Griffiths et al., U.S. Pat. Nos.5,885,793 and 5,969,108; McCafferty et al., WO 92/01047; Liming et al.,WO 99/06587), or domain antibodies using yeast selection system (see,e.g., U.S. Pat. No. 7,195,595). In addition, resurfaced antibodies suchas those disclosed in U.S. Pat. No. 5,639,641 may also be used, as maychimerized or humanized antibodies.

Selection of a particular antigen binding unit is a matter of choicethat depends upon the disease type, cells and tissues that are to betargeted.

In some embodiments, the antigen binding unit is human monoclonalantibody.

Antigen binding units that have specificity to a tumor antigen can beused. A “tumor antigen” as used herein, refers to an antigenic substanceproduced in tumor cells, i.e., it triggers an immune response in thehost. Tumor antigens are useful in identifying tumor cells and arepotential candidates for use in cancer therapy. Normal proteins in thebody are not antigenic. Certain proteins, however, are produced oroverexpressed during tumorigenesis and thus appear “foreign” to thebody. This may include normal proteins that are well sequestered fromthe immune system, proteins that are normally produced in extremelysmall quantities, proteins that are normally produced only in certainstages of development, or proteins whose structure is modified due tomutation.

An abundance of tumor antigens are known in the art and new tumorantigens can be readily identified by screening. Non-limiting examplesof tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47,CD52, CD133, CEA, gpA33, Mucins, TAG-72, CIX, PSMA, folate-bindingprotein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, αVβ3, α5β1, ERBB2, ERBB3,MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.

Hormones and cellular factors can be tumor antigents. Non-limitingexamples include lymphokines such as IL-2, IL-3, IL-4, IL-6, hormonessuch as insulin, thyrotropin releasing hormone (TRH),melanocyte-stimulating hormone (MSH), steroid hormones such as androgensand estrogens; growth factors and colony-stimulating factors such asEGF, TGF-alpha, FGF, VEGF, G-CSF, M-CSF and GM-CSF (see, e.g., Burgess,Immunology Today 5:155-158 (1984)), transferrin (see, e.g., O'Keefe etal., J. Biol. Chem. 260:932-937 (1985)); and vitamins, such as folate.

Antigen binding units having specificity to a protein that isoverexpressed on a tumor cell as compared to a corresponding non-tumorcell can also be used. A “corresponding non-tumor cell” as used here,refers to a non-tumor cell that is of the same cell type as the originof the tumor cell. It is noted that such proteins are not necessarilydifferent from tumor antigens. Non-limiting examples includecarcinoembryonic antigen (CEA), which is overexpressed in most colon,rectum, breast, lung, pancreas and gastrointestinal tract carcinomas;heregulin receptors (HER-2, neu or c-erbB-2), which is frequentlyoverexpressed in breast, ovarian, colon, lung, prostate and cervicalcancers; epidermal growth factor receptor (EGFR), which is highlyexpressed in a range of solid tumors including those of the breast, headand neck, non-small cell lung and prostate; asialoglycoprotein receptor;transferrin receptor; serpin enzyme complex receptor, which is expressedon hepatocytes; fibroblast growth factor receptor (FGFR), which isoverexpressed on pancreatic ductal adenocarcinoma cells; vascularendothelial growth factor receptor (VEGFR), for anti-angiogenesis genetherapy; folate receptor, which is selectively overexpressed in 90% ofnonmucinous ovarian carcinomas; cell surface glycocalyx; carbohydratereceptors; and polymeric immunoglobulin receptor, which is useful forgene delivery to respiratory epithelial cells and attractive fortreatment of lung diseases such as cystic fibrosis.

It is contemplated that antigen binding units can be modified tointroduce an amino acid sequence having improved antibody-dependentcellular cytotoxicity (ADCC). For instance, an IgG2 antibody can bemodified to include an Fc and/or hinge region from an IgG1 antibody toachieve improved ADCC. Examples of IgG1-Fc that mediates improved ADCC,as well as methods of screening for such sequences, are known in the art(e.g., Stewart et al. Protein Eng Des Sel. 24(9):671-8, 2011).

One particular example of such an antigen binding unit, Bat0206, havingimproved ADCC has been tested in the present disclosure. Bat0206includes an anti-EGFR light chain having an amino acid sequence of SEQID NO: 1 and an anti-EGFR heavy chain having an amino acid sequence ofSEQ ID NO: 2. As shown in Table 1, SEQ ID NO NO: 2 includes a fragment(not underlined) derived from an IgG2 antibody which is fused with an Fcfragment of an IgG1 antibody (underlined and bold).

TABLE 1 Amino acid sequences of exemplifying antigen binding unitsSEQ ID NO: Amino acid sequence and name  1Bat0202 (Anti-EGFR Light Chain 1)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC  2Bat0204 (Anti-EGFR heavy Chain 1)QVQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRV TGAFDIWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  3 Cetuximab Light ChainDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGA  4Cetuximab heavy Chain QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK  5EGF-ABXL (Anti-EGFR Light Chain 3)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC  6EGF-ABXH (Anti-EGFR Heavy Chain 3)QLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDERKCCVECPAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  7 Nimotuzumab Light ChainDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITREVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 8 Nimotuzumab Heavy ChainQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYTYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVP  9 Matuzumab Light ChainQVDIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGE 10Matuzumab Heavy Chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS

Another example of the antigen binding unit is Cetuximab which is achimeric (mouse/human) monoclonal antibody targeting epidermal growthfactor receptor (EGFR). The light and heavy chain sequences of Cetuximabare provided as SEQ ID NO: 3 and 4, respectively, shown in Table 1.Another anti-EGFR antibody is Panitumumab which is a fully humanmonoclonal antibody. Still another anti-EGFR antibody is Nimotuzumab,and the light and heavy chain sequences of Nimotuzumab are provided asSEQ ID NO: 7 and 8; still another anti-EGFR antibody is Matuzumab, andthe light and heavy chain sequences of Matuzumab are provided as SEQ IDNO: 9 and 10. EGFR is over expressed in many cancer tissues such asmetastatic colorectal cancer and head and neck cancer.

Rituximab is a chimeric monoclonal antibody against the protein CD20,which is primarily found on the surface of B cells. Rituximabspecifically binds to and destroys B cells, and is therefore used totreat diseases which are characterized by excessive numbers of B cells,overactive B cells, or dysfunctional B cells. This includes manylymphomas, leukemias, transplant rejection, and some autoimmunedisorders. The light chain and heavy chain sequences of Rituximab areprovided in Table 1 as SEQ ID NO: 13 and 14, respectively.

Similarly, the monoclonal antibody trastuzumab (marketed as Herceptin®)is a chimeric monoclonal antibody against the protein Her2, thatinterfering with the Her2/neu receptor. The light chain and heavy chainsequences of trastuzumab are provided in Table 1 as SEQ ID NO: 11 and12, respectively. In some cancers, notably some breast cancers, Her2 isover-expressed, and causes breast cells to reproduce uncontrollably.Trastuzumab is used to treat certain breast cancers and gastric cancers.

Additional examples of antigen binding units and their sequences areprovided in Table 1, without limitation.

Non-antibody molecules can also be used an antigen binding unit totarget specific cell populations. For example, GM-CSF, which binds tomyeloid cells, can be used as a cell-binding agent to target diseasedcells from acute myelogenous leukemia. In addition, IL-2, which binds toactivated T-cells, can be used for prevention of transplant graftrejection, for therapy and prevention of graft-versus-host disease, andfor treatment of acute T-cell leukemia. MSH, which binds to melanocytes,can be used for the treatment of melanoma. Folic acid can be used totarget the folate receptor expressed on ovarian and other tumors.Epidermal growth factor (EGF) can be used to target squamous cancerssuch as lung and head and neck. Somatostatin can be used to targetneuroblastomas and other tumor types. Cancers of the breast and testescan be successfully targeted with estrogen (or estrogen analogues) orandrogen (or androgen analogues) respectively as cell-binding agents.

In some embodiments, the antibody is an equivalent of any one of theantibodies described herein. Equivalents of antibody include thosehaving at least about 80% homology or identity or alternatively, atleast about 85%, or alternatively at least about 90%, or alternativelyat least about 95%, or alternatively 98% homology with nepenthesin, oralternatively a polypeptide or protein encoded by a polynucleotide thathybridizes under stringent conditions to the nucleotide sequenceencoding nepenthesin or its complement, while maintaining the desiredstructure and exhibiting at least part of the antigen binding activityof the antibody.

In another aspect, provided is a D-L-Abu which is one or more ofmaytansinoid lined with Bat0206, wherein Bat0206 comprises an anti-EGFRlight chain having an amino acid sequence of SEQ ID NO: 1 and ananti-EGFR heavy chain having an amino acid sequence of SEQ ID NO: 2, oran equivalent thereof.

In some embodiments, provided herein is a maytansinoid linker Bat0206conjugate of Formula Id or IId:

or a pharmaceutically acceptable salt or solvate thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH; and    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and    -   L^(b) is selected from C₁-C₂₀ alkylene optionally substituted        with C₁-C₄ alkyl, —SO₃H or —P(O)(OH)₂, C₃-C₈ cycloalkylene        optionally substituted with C₁-C₄ alkyl, and C₁-C₂₀ alkylene        which is optionally substituted with C₁-C₄ alkyl —SO₃H or        —P(O)(OH)₂ and wherein one or more of the —CH₂— groups are        independently replaced with C₃-C₈ cycloalkylene, —O—, —S—,        —S—S—, —NR⁸—, —C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂, 3-8        membered heterocycloalkylene optionally substituted with one or        more C₁-C₄ alkyl or oxo.

In some embodiments, L^(b) is C₁-C₂₀ alkylene which is optionallysubstituted with C₁-C₄ alkyl, —SO₃H or —P(O)(OH)₂ and wherein one ormore of the —CH₂— groups are independently replaced with —S—, —S—S—,—C(═O)NR⁸—, —NR⁸C(═O)—,

In some embodiments, the compound of Formula Id is selected from:

or a pharmaceutically acceptable salt or solvate thereof,

In some embodiments, the compound of Formula Id is selected from:

or a pharmaceutically acceptable salt or solvate thereof,wherein

-   -   X is H or Cl;    -   Y is H or methyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁-C₆ alkyl;    -   p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

In some embodiments, the compound of Formula Id is selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Conjugation of a Drug to an Antigen Binding Unit

As discussed, a drug (e.g., a maytansinoid drug derivative) can beconjugated to an antigen binding unit through a linker. In oneembodiment, the antigen binding unit can be modified with appropriatebifunctional modifying agent. In some embodiments, a group comprising athiol (SH) group (also referred to as thio-comprising group) can beintroduced to the side-chain of an amino acid residue, such as theside-chain of a lysine, on the antigen binding unit. For example, theamino group of a lysine residue on the antigen binding unit can beconverted to a thiol-comprising group by reaction with 2-iminothiolane(Traut's Reagent), or with N-succinimidyl 3-(2-pyridyldithio)propanoate(SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), etc andfollowed by reduction with a reducing reagent, such as2-mercaptoethanol, dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine(TCEP).

Non-limiting examples of thiol-comprising group that can replace theside-chain amino group of a lysine residue include —NHC(═NH)(CH₂)_(n)SHand —NHC(O)(CH₂)_(n)SH, wherein n is 1, 2, 3, 4, 5 or 6. When athiol-comprising group is introduced to an amino acid residue, the aminoacid residue is referred to as thiolated amino acid. For example, whenthe side-chain amino group of a lysine residue is converted to athio-comprising group, the lysine residue is referred to as thiolatedlysine. The number of free thiol (SH) group introduced in an antigenbinding unit may vary, such as between 1 and about 20, or 5 to 15, andor 5 to 12. The linkers or drug-linkers can form bonds with the freethiol (SH) group of a thiolated lysine residue on the antigen bindingunit. In some embodiments, the number of linkers or drug-linkers thatform bonds with thiolated lysine residues in the antigen binding unit isbetween 1 and about 10. In some embodiments, the number of such formedbonds is at least 1, or alternatively at least 2, or 3, or 4, or 5. Insome embodiments, the number of such formed bonds is no more than 10, oralternatively no more than 9, or 8, or 7, or 6, or 5, or 4. In someembodiments, each antigen binding unit, on average, is conjugated with3-5 drug molecules.

In another embodiment, a drug-linker can be conjugated to an antigenbinding unit by binding to the thiol group of a cysteine residue. Eachantigen binding unit typically contains multiple cysteines, but many, ifnot all, of them form disulfite bonds between each other, and thus arenot available for such conjugation. In some embodiments, therefore, oneor more of the disufite bonds of the antigen binding unit can be brokento form free thiol (SH) groups by reaction with a reducing reagent, suchas 2-mercaptoethanol, dithiothreitol (DTT) ortris(2-carboxyethyl)phosphine (TCEP), for instance. The reaction can bemonitored and/or controlled so that a sufficient number of disulfitebonds are broken to allow conjugation while maintaining a sufficientnumber of disulfide bonds to keep the structure stability of the antigenbinding unit.

In some embodiments, the number of bonds formed between the drug-linkerand cysteine residue on the antigen binding unit is from 1 to 10. In oneembodiment, the number of such bonds is at least 1, or alternatively atleast 2, or 3, or 4, or 5. In some embodiments, the number of suchformed bonds is no more than 10, or alternatively no more than 9, or 8,or 7, or 6, or 5, or 4. In one embodiment, each antigen binding unit, onaverage, is conjugated with 3-5 drug molecules through cysteines.

In some embodiments, drug molecules are conjugated to the antigenbinding unit through a mixture of lysine and cysteine residues.

An antigen binding unit can be modified, by way of, e.g., site-specificmutagenesis, to introduce additional thiolated lysine or cysteineresidues to allow suitable conjugation. Amino acid modification methodsare well known in the art. Modified antigen binding units can then beexperimentally examined for their stability and antigen bindingcapability. In one embodiment, at least one thiolated lysine or cysteineresidue is introduced by such modification. In another embodiment, atleast two thiolated lysine or cysteine residues are introduced by suchmodification.

Drug Load

The drug load on an antigen binding unit may vary depending on manyfactors, such as the potency of the drug, the size, stability of theantigen binding unit, conjugatable groups available on the antigenbinding unit, etc. In some embodiments, 1 to 10 maytansinoid drugmolecules are conjugated with 1 antigen binding unit molecule. In someembodiments, an average of 3 to 5 maytansinoid drug molecules areconjugated with 1 antigen binding unit molecule. In some embodiments, anaverage of 3.5 maytansinoid drug molecules are conjugated with 1 antigenbinding unit molecule.

Preparation of Drug Linker Antigen Binding Unit Conjugates

In another aspect, provided are methods of preparing a maytansinoid druglinker antigen binding unit conjugate compound comprising contacting acompound of any one of Formula I, I-1, II, II-1, III, III-1, IV, IV-1and V with an antigen binding unit. In some embodiments, the compound ofany one of Formula I, I-1, II, II-1, III, III-1, IV, IV-1 and V and anantigen binding unit are dissolved in an aqueous solution. In someembodiments, the solution comprises a buffer, such as a phosphatebuffer.

Metabolites of Drug Linker Antigen Binding Unit Conjugates

While not wishing to be bound to any theories, it is contemplated thatupon endocytosis, compounds of any one of Formula Ia-Va is degraded byintracellular proteins to degradation products comprising themaytansinoid moiety which are cytotoxic.

In another aspect, disclosed herein is a compound of Formula Ib or Ib-1:

or a salt thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   each Z is independently hydrogen or C₁-C₄ alkyl, or the two Z        with the carbon atom to which they are attached form a C═O;    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR—, —C(O)—, —C(═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; Preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo; and    -   AA is an amino acid, such as a cysteine or a thiolated amino        acid, such as a thiolated lysine.

In some embodiments, the compound is:

or a salt thereof,

In some embodiments, provided herein is a compound of Formula IIb orIIb-1:

or a salt thereof,wherein

-   -   X is hydrogen or halo;    -   Y is selected from the group consisting of hydrogen, C₁-C₆        alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵;    -   R¹ is selected from the group consisting of hydrogen, —OH,        —OC(═O)R⁵ and —OR⁵;    -   R² is hydrogen or C₁-C₆ alkyl;    -   R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶;    -   R⁴ is —OH or —SH;    -   R⁵ is C₁-C₆ alkyl or benzyl;    -   R⁶ is C₁-C₆ alkyl, phenyl or benzyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁₋₆ alkyl;    -   each Z is independently hydrogen or C₁-C₄ alkyl, or the two Z        with the carbon atom to which they are attached form a C═O;    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo; and    -   AA is an amino acid, such as a cysteine or a thiolated amino        acid, such as a thiolated lysine.

In some embodiments, X is hydrogen. In some embodiments, X is chloro. Insome embodiments, Y is hydrogen. In some embodiments, R¹ is hydrogen. Insome embodiments, R² is methyl. In some embodiments, R³ is methyl. Insome embodiments, R⁴ is —OH. In some embodiments, R⁷ is an amino acidside chain. In some embodiments, R⁷ is methyl. In some embodiments, R⁸is methyl.

In some embodiments, L is unsubstituted C₁-C₂₀ alkylene. In someembodiments, L is substituted C₁-C₂₀ alkylene. In some embodiments, L isunsubstituted C₁-C₁₀ alkylene. In some embodiments, L is substitutedC₁-C₁₀ alkylene. In some embodiments, L is —(CH₂)₅—. In someembodiments, L is unsubstituted C₁-C₂₀ alkylene wherein one or two ofthe —CH₂— groups are independently replaced with C₃-C₈ cycloalkylene,—O—, —S—, —NR⁸—, —C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—. In someembodiments, L is substituted C₁-C₂₀ alkylene wherein one or two of the—CH₂— group is independently replaced with C₃-C₈ cycloalkylene, —O—,—S—, —NR⁸—, —C(═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—. In someembodiments, when more than one —CH₂— groups are replaced, the —CH₂—groups are not adjacent to each other.

In some embodiments, provided is a compound of the formula:

In some embodiments, provided is a compound of Formula Mb or IIIb-1:

or a salt thereof,wherein

-   -   X is H or Cl;    -   Y is H or methyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁-C₆ alkyl;    -   L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈        cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein        one or more of the —CH₂— groups are independently replaced with        C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—,        —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; preferably L is —(CH₂)_(m)—,        wherein m is selected from an integer of 1 to 20, preferably 1        to 10, more preferably 5 to 10;    -   substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with        1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is        independently C₁₋₆ alkyl optionally substituted with 1 or 2        substituents independently selected from the group consisting of        —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein        each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,        cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹        together with the nitrogen form a heterocyclic, wherein the        heterocyclic is optionally substituted with one or two oxo; and    -   AA is an amino acid, such as a cysteine or a thiolated amino        acid, such as a thiolated lysine.

In some embodiments, the compound is of Formula IVb or IVb-1:

or a salt thereof,wherein

-   -   X is H or Cl;    -   Y is H or methyl;    -   R⁷ is hydrogen, C₁-C₆ alkyl or an amino acid side chain;    -   R⁸ is hydrogen or C₁-C₆ alkyl;    -   m is an integer of 1 to 20, preferably 1 to 10, more preferably        5 to 10; and    -   AA is an amino acid, such as a cysteine or a thiolated amino        acid, such as a thiolated lysine.

In some embodiments, the compound is of Formula Vb:

or a salt thereof.

In some embodiments, AA is, but not limited to:

wherein

represents point of connection to the rest of the molecule.

In some embodiments, the compound of Formula Ib is selected from:

While not wishing to be bound to any theories, it is contemplated thatupon endocytosis, compounds of any one of Formula Ia-Va, formula Va-XIIais degraded by intracellular proteins to degradation products comprisingthe maytansinoid moiety which are cytotoxic.

In another aspect, disclosed herein is a compound of Formula Vc:

In another aspect, disclosed herein are compounds degraded byintracellular proteins to degradation products comprising themaytansinoid moiety which are cytotoxic, and these compounds include butnot limited to:

Methods of Treatment

In another aspect, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofone or more compounds as described herein, for example, a compound ofany one of Formula Ia-Va, Id and formula Va-XIVc.

The compounds can be formulated as pharmaceutical compositions andadministered to the patient in a variety of forms adapted to the chosenroute of administration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes. The amount of thecompounds will vary depend on the nature of the drug, linker, drug load,degree of cell surface triggered the internalization, trafficking, andrelease of the drug, the disease being treated, the conditions of thepatient, such as age, gender, weight, etc. and can be determined bymethods known to the art, for example, see U.S. Pat. No. 4,938,949, andwill be ultimately at the discretion of the attendant physician orclinician.

In general, a suitable dose will be in the range of from about 0.1 toabout 200 mg/kg, e.g., from about 0.5 to about 50 mg/kg of body weightIV infusion over 30-90 min every 1-4 week for 52 weeks, about 1.0 toabout 25 mg/kg of body weight IV infusion over 30-90 min every 1-4 weekfor 52 weeks, about 1.5 to about 15 mg/kg body weight IV infusion over30-90 min every 1-4 week for 52 weeks, or in the range of about 1 to 10mg/kg body weight IV infusion over 30-90 min every 1-4 week. In someembodiments, the dose is from about 1.0 mg to about 100 mg/day, e.g.,from about 2 mg to about 5 g per day, about 10 mg to about 1 g per day,about 20 to about 500 mg per day, or in the range of about 50 to 100 mgper day. The compounds can be administered daily, weekly, monthly, suchas once a day, every 1-3 weeks, or month. Alternatively, the compoundscan be administered in cycles, such as administered daily for a numberof days, for example, 5 days to 21 days, with a period, such as one dayto seven days, wherein no drug is being administered.

In some embodiments, the compound is administered at an initial dose of1-4 mg/kg over 30-90 minute IV infusion, followed by 1-2 mg/kg over 30minute IV infusion weekly or every 1-4 weeks for 52 weeks. In someembodiments, the compound is administered at an initial dose of 2-10mg/kg over 30-90 minutes IV infusion, followed by 1-5 mg/kg over 30-90minutes IV infusion every 1-4 weeks for 52 weeks.

In some embodiments, the compounds are administered in conjunction withanother therapy. For example, the compounds can be co-administered withanother therapy for treating cancer, for example, radiation therapy oranother anticancer agent known in the art.

In another aspect, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula Ib, wherein the compound of Formula Ib isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula Ia, or a pharmaceuticallyacceptable salt thereof, to the individual. Metabolic chemical reactionrefers to a reaction occurring inside the body, for example, cells, ofthe subject, in which a chemical compound is converted to anotherchemical compound. The conversion can be by metabolic and/or chemicalprocesses and can occur in one step or through a series of two or moresteps. Metabolic chemical reactions include reactions of degrading aprotein or peptide component of a maytansinoid linker antigen bindingunit conjugate, such as an antibody or antibody fragment, by proteinsinside a cell.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula IIb, wherein the compound of Formula IIb isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula IIa, or a pharmaceuticallyacceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula Mb, wherein the compound of Formula IIIb isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula IIIa, or a pharmaceuticallyacceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula VIb, wherein the compound of Formula VIb isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula VIa, or a pharmaceuticallyacceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula Vb, wherein the compound of Formula Vb isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula Va, or a pharmaceuticallyacceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula Vc, wherein the compound of Formula Vc isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula Va, VIa, or VIIa, or apharmaceutically acceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula VIIIc, wherein the compound of Formula VIIIc isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula VIIIa, or a pharmaceuticallyacceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula IXc, wherein the compound of Formula IXc isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula IXa, or a pharmaceuticallyacceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula Xc, wherein the compound of Formula Xc isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula Xa, or a pharmaceuticallyacceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula XIc, wherein the compound of Formula XIc isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula XIa, or a pharmaceuticallyacceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula XIIc, wherein the compound of Formula XIIc isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula XIIa, or a pharmaceuticallyacceptable salt thereof, to the patient.

In some embodiments, provided herein is a method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula XIVc, wherein the compound of Formula XIVc isgenerated as a result of a metabolic chemical reaction followingadministration of a compound of Formula XIVa, or a pharmaceuticallyacceptable salt thereof, to the patient.

The diseases being treated can be determined by the antigen binding unitof the conjugate. In some embodiments, the disease a proliferativedisease, such as a cancer, including melanoma, breast cancer, bladdercancer, lung cancer, thyroid cancer, prostate cancer, ovarian cancer,mast cell leukemia, germ cell tumors, small-cell lung carcinoma,gastrointestinal stromal tumors, acute myelogenous leukemia (AML),B-chronic lymphatic leukemia (B-CLL), and non-Hodgkin lymphoma (NHL),neuroblastoma, or pancreatic cancer. In some embodiments, the disease aninflammatory disease, or an immunologic disease, such as graftrejections, and autoimmune diseases, such as type 1 diabetes, rheumatoidarthritis, systemic lupus erythematosus, and inflammatory bowel disease.

Pharmaceutical Compositions

In a further aspect, provided are pharmaceutical compositions comprisingone or more compounds as described herein, for example, a compound ofany one of Formula Ia-Va, and one or more pharmaceutically acceptablecarriers. Such compositions should contain at least 0.1% of activecompound. The percentage of the compositions may vary and may be betweenabout 2 to about 90% of the weight of a given unit dosage form. Theamount of active compound in such therapeutically useful compositions issuch that an effective dosage level will be obtained.

Examples of compositions for oral administration include, but are notlimited to, ingestible tablets, buccal tablets, troches, capsules,elixirs, suspensions, syrups, solutions, wafers, and the like.Compositions suitable for injection or infusion can include sterileaqueous solutions or dispersions in a pharmaceutically acceptable liquidcarrier or vehicle, or sterile powders comprising the active ingredientwhich are adapted for the extemporaneous preparation of sterileinjectable or infusible solutions or dispersions, optionallyencapsulated in liposomes. Other forms of pharmaceutical compositionsinclude topical formulations, such as gel, ointments, creams, lotions ortransdermal patches, etc.

The pharmaceutical compositions include using techniques well known tothose in the art. Suitable pharmaceutically-acceptable carriers, outsidethose mentioned herein, are known in the art; for example, seeRemington, The Science and Practice of Pharmacy, 20th Edition, 2000,Lippincott Williams & Wilkins, (Editors: Gennaro, A. R., et al.).

In a further aspect, provided are methods of producing a pharmaceuticalcomposition comprising admixing a compound as described herein, forexample, a compound of any one of Formula Ia-Va and Id, and apharmaceutically acceptable carrier. Methods of admixing an activeingredient with a pharmaceutically acceptable carrier are generallyknown in the art, for example, uniformly mixing the active compound(s)with liquids or finely divided solid carriers, or both, in the requiredproportions, and then, if necessary, forming the resulting mixture intoa desired shape.

In some embodiments, a compound of any one of Formula Ia-Va and Id isformulated as an injectable, for example, at a concentration of 2-50mg/mL in an aqueous solution comprising 4-10 mg/mL sodium chlorideand/or 5-12 mg/mL sodium acetate, or alternatively at a concentration of2-50 mg/mL in an aqueous solution comprising 5-10 mg/mL sodium chloride,1-5 mg/mL sodium phosphate dibasic heptahydrate, 0.1-0.5 mg/mL sodiumphosphate monobasic monohydrate.

Other examples of formulations of a compound of any one of Formula Ia-Vaand Id include an injectable formulation having a concentration of 2-100mg/mL of the compound in an aqueous solution comprising 0.5-1.0% sodiumchloride, 0.05-0.10% monobasic sodium phosphate dihydrate, 1.0-2.0%dibasic sodium phosphate dihydrate, 0.01-0.05% sodium citrate,0.10-0.20% citric acid monohydrate, 1.0-2.0% mannitol, 0.1%-0.2polysorbate 80, and Water for Injection, USP. Sodium hydroxide added asnecessary to adjust pH.

Methods

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc) are given, other process conditionscan also be used unless otherwise stated. Optimum reaction conditionsmay vary with the particular reactants or solvent used, but suchconditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

Furthermore, the compounds of this invention may contain one or morechiral centers. Accordingly, if desired, such compounds can be preparedor isolated as pure stereoisomers, i.e., as individual enantiomers ordiastereomers, or as stereoisomer-enriched mixtures. All suchstereoisomers (and enriched mixtures) are included within the scope ofthis invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents and the like.

The starting materials for the following reactions are generally knowncompounds or can be prepared by known procedures or obviousmodifications thereof. For example, many of the starting materials areavailable from commercial suppliers such as Aldrich Chemical Co.(Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka-Chemce orSigma (St. Louis, Mo., USA). Others may be prepared by procedures, orobvious modifications thereof, described in standard reference textssuch as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15(John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds,Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989),Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March'sAdvanced Organic Chemistry, (John Wiley and Sons, 4^(th) Edition), andLarock's Comprehensive Organic Transformations (VCH Publishers Inc.,1989).

The various starting materials, intermediates, and compounds of theinvention may be isolated and purified where appropriate usingconventional techniques such as precipitation, filtration,crystallization, evaporation, distillation, and chromatography.Characterization of these compounds may be performed using conventionalmethods such as by melting point, mass spectrum, nuclear magneticresonance, and various other spectroscopic analyses.

Coupling reagents include carbodiimide, amininum and phosphonium basedreagents. Carbodiimide type reagents include dicyclohexylcarbodiimide(DCC), diisopropylcarbodiimide (DIC), and1-ethyl-3-(3-dimethylaminopropyl)-dicarbodiimide (EDC), etc. Aminiumsalts includeN-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HATU),N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HBTU),N-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (TCTU),N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumtetrafluoroborate N-oxide (TBTU), andN-[(1H-6-chlorobenzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumtetrafluoroborate N-oxide (TCTU). Phosphonium salts include7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate (PyAOP) andbenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP). Amide formation step may be conducted in a polar solvent suchas dimethylformamide (DMF) and may also include an organic base such asdiisopropylethylamine (DIEA) or dimethylaminopyridine (DMAP).

The following examples are provided to illustrate certain aspects of thepresent invention and to aid those of skill in the art in practicing theinvention. These examples are in no way to be considered to limit thescope of the invention.

EXAMPLES

The examples below as well as throughout the application, the followingabbreviations have the following meanings. If not defined, the termshave their generally accepted meanings.

-   -   ACN=acetonitrile    -   Ala=alanine    -   aq.=aqueous    -   brs=broad singlet    -   calc.=calculated    -   d=doublet    -   DCM=dichloromethane    -   dd=double doublet    -   DIEA=N,N-diisopropylethylamine    -   DMA=N,N-dimethylacetamide    -   DMAP=dimethylaminopyridine    -   DMF=dimethylformamide    -   DMSO=dimethylsulfoxide    -   DTT=dithiothreitol    -   EDTA=ethylenediaminetetraacetate or salt    -   Et=ethyl    -   EtOAc=ethyl acetate    -   g=gram    -   h=hour    -   HCl=hydrochloric acid    -   HPLC=high-pressure liquid chromatography    -   Hz=hertz    -   J=coupling constant    -   LC-MS=liquid chromatography mass spectroscopy    -   m=multiplet    -   MDC=maytansinol    -   Me=methyl    -   MeOH=methanol    -   MHz=megahertz    -   min=minute    -   mL=milliliter    -   mm=millimeter    -   m.p.=melting point    -   OTf=triflate (trifluoromethanesulfonate)    -   N=normal    -   r.t.=room temperature    -   PBS=phosphate buffered saline    -   Rt=retention time    -   s=singlet    -   t=triplet    -   TLC=thin layer chromatography    -   vol=volume    -   μL=microliter    -   μm=micrometer

MATERIALS AND METHODS: NMR specetra were recorded on a Bruker AM 400(400 MHz) spectrometer. Chemical shifts in CDCl₃ are reported in ppmrelative to residual CHCl₃ as an internal standard. UV spectra wererecorded on a Beckman DU-640 spectrophotometer. Mass spectra wereacquired on a ThermoFinnigan LCQ DECA XP+ instrument using electrosprayionization. HPLC was performed using an Agilent HPLC 1100 systemequipped with a diode array detector and a Kromasil reverse phase C-18 5μm, 250×4.6 mm column, eluting with a gradient of acetonitrile:water(50-95% CH₃CN 0-10 min, 95% CH₃CN 10-15 min, flow rate=1.0 mL/min.Silica gel for flash column chromatography was from Branch of QiangdaoHaiyang Chemical Co., Ltd. Maytansinol was prepared from ansamitocin P-3(which in turn was obtained from the fermentation of the microorganismActinosynnema pretiosum) as previously described (Widdison, et al.(2006) J. Med. Chem. 49: 4392-4408). Dichloromethane was dried bydistillation over calcium hydride. Dimethylformamide was dried bydistillation over calcium hydride under reduced pressure. All othersolvents used are reagent grade or HPLC grade.

Example 1 Esterification of Maytansinol with Fmoc-N-methyl-L-alanine(Fmoc-N-Me-D/L-Ala-MDC)

A mixture of maytansinol (0.600 g, 1.062 mmol), Fmoc-N-Me-L-Ala (6.911g, 21.24 mmol), Sc(OTf)₃ (0.314 g, 0.637 mmol) and DMAP (0.389 g, 3.186mmol) in CH₂Cl₂ (100 mL) was stirred for 0.5 h at −8° C. DIC (2.949 g,23.37 mmol) was added dropwise, stirred for 0.5 h, warmed to r.t.slowly, filtered to recover the Lewis acid catalyst, the filtrate wasquenched with diluted HCl and extracted with CH₂Cl₂. The combinedorganic phase was washed with NaHCO₃ aq, brine, dried over anhydrousNa₂SO₄. The solvent was removed under reduced pressure. Chromatography(silica gel, CH₂Cl₂/MeOH 30:1) gave the desired product as a mixture ofdiastereomer Fmoc-N-Me-D/L-Ala-MDC: white solid (0.8385 g, 90.5%).Further column chromatography (silica gel, CH₂Cl₂/MeOH 100:1 to 20:1)gave two fractions as pure diastereomer. The higher Rf fraction wasdetermined to be the D-aminoacyl ester diastereomer(Fmoc-N-Me-D-Ala-MDC), while the lower Rf fraction was the desiredL-aminoacyl ester (Fmoc-N-Me-L-Ala-MDC). Fmoc-N-Me-L-Ala-MDC: whitesolid (0.4262 g, 46.0% yield), ¹H NMR (400 MHz, CDCl₃): δ0.77 (3H, s),1.22-1.32 (6H, m), 1.40-1.48 (1H, m), 1.63 (3H, s), 2.13 (1H, dd,J=14.4, 2.8 Hz), 2.53 (1H, dd, J=14.4, 10.8 Hz), 2.64 (3H, s), 2.88 (3H,s), 3.00 (1H, d, J=9.6 Hz), 3.07 (1H, d, J=12.4 Hz), 3.35 (3H, s), 3.48(1H, d, J=8.8 Hz), 3.59 (1H, d, J=11.2 Hz), 3.97 (3H, s), 4.13-4.19 (1H,m), 4.15 (1H, s), 4.24 (1H, t, J=10.8 Hz), 4.72-4.77 (2H, m), 5.03 (1H,q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.29 (1H, br), 6.41 (1H,dd, J=15.2, 11.2 Hz), 6.52 (1H, d, J=1.2 Hz), 6.70 (1H, d, J=10.8 Hz),6.79 (1H, d, J=1.2 Hz), 7.33 (1H, t, J=7.6 Hz), 7.36 (1H, t, J=7.6 Hz),7.39 (1H, d, J=7.6 Hz), 7.49 (1H, d, J=7.6 Hz), 7.70 (1H, d, J=7.6 Hz),7.72 (1H, d, J=7.6 Hz). LC-MS (M+Na⁺) calc.: 894.3. found: 894.3.Fmoc-N-Me-D-Ala-MDC: white solid (0.3993 g, 43.1% yield), ¹H NMR (400MHz, CDCl₃): δ0.84 (3H, s), 1.22-1.27 (3H, m), 1.40-1.48 (1H, m), 1.51(3H, d, J=7.6 Hz), 1.67 (3H, s), 2.20 (1H, dd, J=14.4, 2.8 Hz), 2.63(1H, dd, J=14.4, 12.4 Hz), 2.85 (1H, d, J=9.6 Hz), 2.96 (3H, s), 3.17(3H, s), 3.20 (1H, s), 3.24 (3H, s), 3.40 (1H, d, J=9.2 Hz), 3.51 (1H,d, J=12.8 Hz), 3.99 (3H, s), 4.20-4.28 (2H, m), 4.38-4.43 (2H, m),4.80-4.98 (2H, m), 5.80 (1H, dd, J=15.2, 11.2 Hz), 6.18 (1H, s), 6.25(1H, d, J=10.8 Hz), 6.40 (1H, dd, J=15.2, 11.2 Hz), 6.79 (1H, d, J=1.6Hz), 6.84 (1H, d, J=1.6 Hz), 7.32 (2H, t, J=7.6 Hz), 7.41 (2H, t, J=7.6Hz), 7.61 (2H, d, J=7.6 Hz), 7.77 (2H, d, J=7.6 Hz). LC-MS (M+Na⁺)calc.: 894.3. found: 894.3.

Example 2 Deprotection of Fmoc-N-Me-D/L-Ala-MDC (N-Me-D/L-Ala-MDC)

Into Fmoc-N-Me-D/L-Ala-MDC (0.463 g, 0.5307 mmol) in ACN (200 mL) wasadded piperidine (0.865 g, 10.15 mmol). The mixture was stirred at r.t.for 4 h, quenched with water and extracted with CH₂Cl₂. The combinedorganic phase was washed with brine and dried over Na₂SO₄. The solventwas removed under reduced pressure to give the crude product, which wasused in the next step without further purification. LC-MS (M+H⁺) calc.:650.3. found: 650.3. Rt: 3.96 min.

Example 3 Deprotection of Fmoc-N-Me-L-Ala-MDC (N-Me-L-Ala-MDC)

Into Fmoc-N-Me-L-Ala-MDC (0.463 g, 0.5307 mmol) in ACN (200 mL) wasadded piperidine (0.865 g, 10.15 mmol). The mixture was stirred at r.t.for 4 h, quenched with water and extracted with CH₂Cl₂. The combinedorganic phase was washed with brine and dried over Na₂SO₄. The solventwas removed under reduced pressure to give the crude product, which wasused in the next step without further purification. LC-MS (M+H⁺) calc.:650.3. found: 650.3. Rt: 3.96 min.

Example 4 Condensation of N-Me-D/L-Ala-MDC with MA-ACP (D-3AA-MDC andL-3AA-MDC)

Into above prepared N-Me-D/L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g,2.123 mmol) in DMF (25 mL) under 0° C. was added EDC (0.407 g, 2.123mmol). The mixture was stirred at r.t. overnight, quenched with water,extracted with EtOAc, washed with brine, and dried over Na₂SO₄. Thesolvent was removed under reduced pressure. Chromatography (silica gel:CH₂Cl₂/MeOH 30:1) gave the crude product. Further purification bypreparative HPLC on a YMC C-18 column (250×20 mm, S 10 μm) gave twofractions (Rt=6.59 min and 6.98 min) as white solid. The higher Rtfraction was determined to be the D-aminoacyl ester diastereomer(D-3AA-MDC, 45.2%), while the lower Rt fraction was the desiredL-aminoacyl ester (L-3AA-MDC, 54.8%). L-3AA-MDC: white solid (0.1364 g,30.5% overall yield over two steps), ¹H NMR (400 MHz, CDCl₃): δ0.79 (3H,s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m),2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H,s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34(3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d,J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H,s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72(1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na⁺) calc.: 865.3. found:865.3. Rt: 6.59 min. D-3AA-MDC: white solid (0.1128 g, 25.2% overallyield over two steps), ¹H NMR (400 MHz, CDCl₃): δ0.86 (3H, s), 1.22-1.38(4H, m), 1.25 (3H, d, J=9.2 Hz), 1.38-1.45 (1H, m), 1.48 (3H, d, J=7.6Hz), 1.56-1.70 (4H, m), 1.68 (3H, s), 1.75 (1H, d, J=13.6 Hz), 2.19 (1H,dd, J=14.4, 2.8 Hz), 2.28-2.36 (2H, m), 2.65 (1H, dd, J=14.2, 12.0 Hz),2.80 (1H, d, J=9.6 Hz), 3.01 (3H, s), 3.19 (1H, d, J=13.2 Hz), 3.32 (3H,s), 3.42 (1H, d, J=9.6 Hz), 3.47-3.54 (3H, m), 3.98 (3H, s), 4.29 (1H,t, J=10.4 Hz), 4.88 (1H, dd, J=11.8, 3.2 Hz), 5.07 (1H, q, J=7.6 Hz),5.84 (1H, dd, J=15.2, 9.2 Hz), 6.23 (1H, d, J=11.2 Hz), 6.27 (1H, s),6.41 (1H, dd, J=15.2, 11.2 Hz), 6.69 (2H, s), 6.79 (1H, d, J=1.2 Hz),6.84 (1H, d, J=1.2 Hz). LC-MS (M+Na⁺) calc.: 865.3. found: 865.3. Rt:6.98 min.

Example 5 Condensation of N-Me-L-Ala-MDC with MA-ACP (L-3AA-MDC)

Into above prepared N-Me-L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g,2.123 mmol) in DMF (25 mL) under 0° C. was added EDC (0.407 g, 2.123mmol). The mixture was stirred at r.t. overnight, quenched with water,extracted with EtOAc, washed with brine, dried over Na₂SO₄. The solventwas removed under reduced pressure. Chromatography (silica gel:CH₂Cl₂/MeOH 30:1) gave the crude product. Further purification bypreparative HPLC on a YMC C-18 column (250×20 mm, S 10 μm) gave thedesired L-3AA-MDC: white solid (0.280 g, 62.6% overall yield over twosteps), ¹H NMR (400 MHz, CDCl₃): δ0.79 (3H, s), 1.17-1.32 (3H, m), 1.27(3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45(1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz),3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27(1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz),5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s).LC-MS (M+Na⁺) calc.: 865.3. found: 865.3. Rt: 6.59 min.

Example 6 The Effect of the Metabolites of Prodrug Antibody MaytansinoidConjugates on the Tubulin Polymerization

The effect of 3AA-MDC, 206-3AA-MDC and the metabolites (Cys-3AA-MDC andLys-mcc-MDC) of prodrug antibody maytansinoid conjugates on the tubulinpolymerization in vitro was assessed by HTS-Tubulin Polymerization AssayKit (BK004P, Cytoskeleton, Inc., USA). According to the instruction ofkit, pre-warm the 96-well plate to 37° C. for 30 min prior to startingthe assay. At the same time, the spectrophotometer (SpectraMax,Molecular Devices, USA) was set as follow: wavelength, 405 nm;temperature, 37° C.; Kinetic, 31 cycles of 1 reading per minute. Makecold G-PEM buffer (990 μL General Tubulin Buffer+10 μL GTP Stock) andkeep it on ice. Prepare 4 mg/mL tubulin, 1 μM L-3AA-MDC(N₂′-deacetyl-N₂′-(6-maleimido-1-oxo-hexyl)maytansine), 1 μM206-3AA-MDC, 1 μM cys-3AA-MDC, 1 μM lys-mcc-MDC, 100 μM Paclitaxel, and100 μM Nocodazole. Add 10 μL G-PEM, 3AA-MDC, 206-3AA-MDC, cys-3AA-MDC,lys-mcc-MDC, Paclitaxel, Nocodazole into the wells, and then add 100 μL4 mg/ml tubulin to each well. Immediately place the plate into thespectrophotometer and start recording using the kinetic setup describedabove. As show in the FIG. 6, compared with the PBS buffer, 3AA-MDC,Cys-3AA-MDC, Lys-mcc-MDC and 206-3AA-MDC more significantly inhibitedthe tubulin polymerization (FIG. 6). Nocodazole, the tubulinpolymerization inhibitor, was set as a negative control. The metaboliteCys-3AA-MDC was prepared by reaction of 3AA-MDC with cysteine under thebase DIEA in CH₂Cl₂. LC-MS (M+H⁺) calc.: 964.5. found: 964.2. Rt: 12.97min. The metabolite Lys-MCC-MDC was prepared by reaction of SMCC-MDCwith lysine under the base DIEA in DMF. LC-MS (M+H⁺) calc.: 1103.7.found: 1103.2. Rt: 13.00 and 13.18 min.

Example 7 Recombinant Antibody Expression and Purification

The monoclonal antibodies, Bat0206, Bat0606 (trastuzumab), Bat1206(rituximab), which specifically bind the extracellular domain of EGFR,Her2, CD20 respectively were produced as described in CHO cellsessentially as described in Wood et al., J Immunol. 145:3011 (1990).Briefly, each of the antibody genes were constructed with molecularbiology techniques (Molecular Cloning: A Laboratory Manual, 3^(rd)edition J. Sambrook et al., Cold spring Harbor Laboratory Press). Aderivative of Chinese hamster ovary cell lines CHOK1 was grown in CD-CHOmedia (GIBCO). Transfections were facilitated using electroporation.Healthy mid-log CHO-K1 cells were pelleted by centrifuge and wereresuspended in fresh CD-CHO media to achieve cell densities ofapproximately 1×10⁷ cells (600 mL) per cuvette. Suspensions of cellscontaining 40 μg of linearized plasmid DNA were electroporated, seeding10³ cells per well in 96-well tissue culture plates containing suitableselection drug. The antibody expression level in the culture supernatantof clones isolated on 96-well tissue culture plates was determined by anenzyme-linked immunosorbent assay (ELISA). On the basis of the antibodytiter in the supernatant, clones with high-level expression weretransferred to 24-well plate (Corning) containing suitable media.Specific antibody productivity (qAb) and specific growth rate (μ) werefurther analyzed by seeding cells at 2×10⁵ cells per well containing 5ml of medium in six-well tissue culture plates, culturing for 2 and 4days, and usually 20-30 high-producing clones (parental clones) weretransferred to shake flask for successive selection, and 5-8 highestproducer clones were chosen to be further subcloned, and tested forexpression.

The purification was carried out by centrifuging cell suspension andharvesting the supernatant, which was further cleared by centrifuging.Protein A affinity columns such as Mab Select SuRe (GE Healthcare) andion exchange such as Capto S (GE) were used to purify the expressedantibodies).

Example 8 Conjugation of Bat0206 with SMCC-MDC

The drug-linker SMCC-MDC was prepared in the following reactions: (1)3-mercaptopropanoic acid (MPr) was reacted with N-succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC) in the presence ofDIEA, giving the MPr-SMCC at a yield of over 95%; Secondly, condensationof N-Me-L-Ala-MDC, which was prepared by deprotection ofFmoc-N-Me-Ala-MDC under a base piperidine in CH₃CN, with MPr-SMCC undera coupling reagent EDC, giving the desired coupled product SMCC-MDC in60-70% yield over two steps. Antibody Bat0206 (Abu) was diluted to 2.5mg/mL in solution A (50 mM potassium phosphate, 50 mM NaCl, and 2 mMEDTA, pH 6.5). SMCC-MDC was added to give a ratio of SMCC-MDC toantibody of 7:1 mole equivalent. Then DMA was added to 15% (v/v) to thereaction and reaction was mixed by stirring for 4 h at ambienttemperature. D-Lmcc-Bat0206 conjugate was purified from excess unreactedor hydrolyzed reagent and excess SMCC-MDC using a G25 gel filtrationcolumn equilibrated in pH 7.4 phosphate buffer (aqueous). The conjugatewas then dialyzed overnight into pH 7.4 phosphate buffer (aqueous) andthen filtered through a 0.22 μm filter for final storage. The number ofSMCC-MDC molecule per Abu molecule in the final conjugate was measuredby determining absorbance of the conjugate at 252 and 280 nm and usingknown extinction coefficients for SMCC-MDC and antibody at these twowavelengths. A ratio of maytansinoid compound to antibody of 3.5:1.0 wasnormally obtained.

Example 9 Conjugation of Bat0206 with Batansine

Antibody Bat0206 was diluted to 8.0 mg/mL in solution B (50 mM potassiumphosphate, 50 mM NaCl, and 2 mM EDTA, pH 8.0). Partial reduction wascarried out with (6 moles equivalent) DTT. After incubation at 37° C.for 60 minutes, the buffer was exchanged by elution through SephadexG-25 resin with solution B. The thiol-antibody value was determined fromthe reduced monoclonal antibody (mAb) concentration determined from280-nm absorbance, and the thiol concentration was determined byreaction with DTNB (5,5′-dithiobis(2-nitrobenzoic acid); Aldrich) andabsorbance measured at 412 nm.

The conjugation reaction was carried out with 10% DMA. The batansine(3AA-MDC) was prepared as in Example 4 and 5 above. The volume ofbatansine solution was calculated to contain 1.5-mol batansine (3AA-MDC)per mol thiol equivalent. Batansine solution was added rapidly withmixing to the cold-reduced antibody solution, and the mixture wasstirred at r.t. for 3 hours, and continued for additional 1 h afteradding 5 mM cysteine. The reaction mixture was concentrated bycentrifugal ultrafiltration and buffer-exchanged by elution throughSephadex G25 equilibrated in PBS. The conjugate was then filteredthrough a 0.2-μm filter under sterile conditions and stored at −80° C.for analysis and testing. The Batansine-0206 was further analyzed fordrug/antibody ratio by measuring unreacted thiols with DTNB, and 3.5:1ratio of drug/antibody was often obtained. Batansine-0206 was furthercharacterized for concentration by UV absorbance, aggregation bysize-exclusion chromatography, and residual free drug by reverse-phaseHPLC. All mAbs and ADCs used in these studies exceeded 98% monomericprotein.

Example 10 Characterization of Batansine-0206

The growth inhibitory characteristics of Bat0206 and Batansine-0206 wereevaluated using the EGFR positive breast tumor cell line, MDA-MB-468 andEGFR negative cell line BT474 (Shanghai Cell Collections, Ltd. Co.,Shanghai, China). Briefly, cells were detached by using 0.25% (vol/vol)trypsin and suspended in complete medium. Aliquots of 100 μL containing10,000 cells were plated into 96-well microplates. The cells wereallowed to adhere overnight at 37° C., and 100 μL of media containingvarious concentrations of Bat0206 and Batansine-0206 was then added.After 72 hours, plates were washed twice with PBS (pH 7.5), and analyzedfor relative cell proliferation with Cell Counting Kit-8 (CCK-8, DojindoMolec. Technologies, Japan) reagent. Drug conjugate Batansine-0206significantly inhibited the EGFR positive cell proliferation at muchlower concentration than naked Bat0206 and naked Cetuximab (FIG. 7).Neither naked antibodies Bat0206, naked marked antibody Cetuximab, nordrug conjugate Batansine-0206 inhibited the growth of EGFR negative cellline BT474 (FIG. 8).

Example 11 Preparation of Antibody-Drug Conjugates: D-Lmcc-Bat0206

Antibody Bat0206 was diluted to 2.5 mg/mL in solution A (50 mM potassiumphosphate, 50 mM NaCl, and 2 mM EDTA, pH 6.5). SMCC-MDC was added togive a ratio of SMCC-MDC to antibody of 7:1 mole equivalent. Then DMAwas added to 15% to the reaction and reaction was mixed by stirring for4 h at ambient temperature. D-Lmcc-Bat0206 conjugate was purified fromexcess unreacted or hydrolyzed reagent and excess SMCC-MDC using a G25gel filtration column equilibrated in pH 7.4 phosphate buffer (aqueous).The conjugate was then dialyzed overnight into pH 7.4 phosphate buffer(aqueous) and then filtered through a 0.22 μm filter for final storage.The number of SMCC-MDC molecule per Abu molecule in the final conjugatewas measured by determining absorbance of the conjugate at 252 and 280nm and using known extinction coefficients for SMCC-MDC and antibody atthese two wavelengths. A ratio of maytansinoid compound to antibody of2-5 to 1 was normally obtained.

The growth inhibitory characteristics of D-Lmcc-Bat0206 were alsoevaluated using the EGFR positive breast tumor cell line, MDA-MB-468 andEGFR negative cell line BT474. Briefly, cells were detached by using0.25% (vol/vol) trypsin and suspended in complete medium. Aliquots of100 μL containing 10,000 cells were plated into 96-well microplates. Thecells were allowed to adhere overnight at 37° C., and 100 μL of mediacontaining various concentrations of Bat0206 and D-Lmcc-bat0206 was thenadded. After 72 hours, plates were washed twice with PBS (pH 7.5), andanalyzed for relative cell proliferation with CCK-8 reagent. Drugconjugate D-Lmcc-Bat0206, as in the case of Batansine-0206,significantly inhibited the EGFR positive cell proliferation at muchlower concentration than naked Bat0206 and naked Cetuximab (FIG. 10).

Example 12 D-Lmcc-Bat0206 Eradicates Human A431 Tumor Xenografts

In Vivo Tumor Studies: The effects of D-Lmcc-Bat0206 on the growth ofestablished tumors were examined on human A431 tumor xenografts. HumanA431 cells (ATCC, CRL-7907) were cultured in DMEM medium supplementedwith 10% fetal bovine serum, 2 mM glutamine and antibiotics. FemaleBALB/c nude mice, 4-6 weeks old, were injected subcutaneously with 4×10⁶tumor cells in the dorsal area in a volume of 100 μL. When the tumorxenografts reaches a size of 80-200 mm³ (calculated as 0.5(length×width²), animals were then treated with Bat0206, D-Lmcc-Bat0206,or a control buffer. Bat0206 and D-Lmcc-Bat0206 were administered at thedoses of 5 mg/kg. Animals were dosed every 3 days for a total of 8 dosesi.p. in a volume of 100 μL. Each group consisted of 12 mice. Tumor sizewas determined at 3 days intervals. Twenty four days after tumor cellinoculation, animals were euthanized and tumors were removed andweighed. As shown in FIGS. 11 and 12, at 5 mg/kg dose tested, Bat0206and D-Lmcc-Bat0206 markedly suppressed tumor growth as assessed by tumorweight measurements 24 days after drug treatment.

Example 13 Preparation of Antibody-Drug Conjugate D-LSPP-Bat0206

Antibody Bat0206 (8 mg/mL) was modified using 8-fold molar excess ofN-succinimidyl-4-(2-pyridyldithio) pentanoate (SPP) to introducedithiopyridyl groups. The reaction was carried out in 95% v/v Buffer A(50 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5) and 5% v/vDMA for 2 h at room temperature. The slightly turgid reaction mixturewas gel-filtered through a Sephadex G25 column (equilibrated in BufferA). The degree of modification was determined by measuring theabsorbance of the antibody and the 2-mercaptopyridine (Spy) released byDTT respectively at 280 and 343 nm. Modified Bat0206 was then conjugatedat 2.5 mg/mL using a 1.7-fold molar excess ofN₂′-deacetyl-N₂′-(3-mercapto-1-oxopropyl)-maytansine over SPy. Thereaction was carried out with DMA (5% v/v) in Buffer A (see above). Thereaction was incubated at room temperature overnight for 17 h. Theconjugated antibody was cleared by centrifugation and then furtherpurified through gel-filteration with a Sephadex G25 column equilibratedwith PBS pH 6.5. The conjugate was sterile-filtered using a 0.22 μMMillex-GV filter. The number of drug molecules linked per Bat0206molecule was determined by measuring the absorbance at both 252 nm and280 nm of the filtered material. The drug to antibody ratio was found tobe about 4.5. The conjugated antibody was further biochemicallycharacterized by size exclusion chromography (SEC) and found to be over96% monomer.

The growth inhibitory characteristics of D-Lspp-Bat0206 were evaluatedusing the EGFR positive tumor cell line A431 and A549. Briefly, cellswere detached by using 0.25% (vol/vol) trypsin and suspended in completemedium. Aliquots of cells were plated into 96-well microdilution plates.The cells were allowed to adhere overnight at 37° C., and 100 μL ofmedia containing various concentrations of Bat0606 and D-Lspp-Bat0206was then added. After 72 hours, plates were washed twice with PBS (pH7.5), and analyzed for relative cell proliferation with CCK-8 reagent.Drug conjugate D-Lspp-Bat0206 significantly inhibited the A431 positivecell proliferation at much lower concentration than naked Bat0206 (FIG.13), and furthermore, D-Lspp-bat0206 effectively inhibited the growth ofA549 cells, which is resistant to treatment by naked Bat0206 as a resultof Kras mutation (FIG. 14).

The IC₅₀ values of certain compounds are presented in Tables 1 and 2.The data in Table 2 are obtained with conjugates having different drugloads and using different experimental conditions.

Batansine-0206 and D-Lmcc-Bat0206 significantly increased the activityof the naked antibody against EGFR positive tumor cell lines. Comparedwith the di-sulfide comprising drug-linker-antibody D-Lspp-Bat0206,Batansine-0206 and D-Lmcc-Bat0206 exhibited significant improvement inselectivity against EGFR positive tumor cell lines A431 and MDA-MB-468.Surprisingly, Batansine-0206 and D-Lmcc-Bat0206 also exhibitedsignificant improvement in selectivity against EGFR positive tumor celllines A431 and MDA-MB-468 over the naked antibody.

TABLE 1 IC₅₀ IC₅₀ IC₅₀ IC₅₀ D-Lmcc- D-Lspp- Batansine- Cell line Bat0206Bat0206 Bat0206 0206 A431 >100 nM   22 nM  2.9 nM 0.17 nM MDA-MB-468 0.46 nM  0.15 nM 0.10 nM 0.06 nM HCT116 >100 nM 53.24 nM 0.96 nM 44.22nM 

TABLE 2 IC₅₀ IC₅₀ IC₅₀ Cell line Bat0606 D-Lmcc-Bat0606 Batansine-0606Her2 positive breast 0.287 nM 0.099 nM 0.041 nM tumor cell line SK-BR-3EGFR negative Her2- 0.791 nM 0.257 nM 0.474 nM positive breast cancercells BT474 Her2-overexpressing 0.221 nM 0.139 nM human gastric cancercell line NCI-N87

Example 14 Batansine-0606 Eradicates Human BT474 Tumor Xenografts

In Vivo Tumor Studies: The effects of batansine-0606(3AA-MDC-trastuzumab) on the growth of established tumors were examinedon human BT474 tumor xenografts. Human BT474 cells (ATCC, HTB-20) werecultured in DMEM medium supplemented with 10% fetal bovine serum, 2 mMglutamine and antibiotics. Female BALB/c nude mice, 8-9 weeks old, wereinjected subcutaneously with 1×10⁷ tumor cells in the dorsal area in avolume of 100 μL. When the tumor xenografts reaches a size of 150-200mm³ (calculated as 0.5× (length×width), animals were then treated withBat0606 (5 or 15 mg/kg), batansine-0606 (5 or 15 mg/kg), or controlantibody (Rituximab, 15 mg/kg). Animals were dosed every 3 weeks for atotal of 3 doses i.v. in a volume of 100 μL. Each group consisted of 10mice. Tumor size was determined at 3 days intervals. 49 days after tumorcell inoculation, animals were euthanized and tumors were removed andweighed. As shown in FIG. 30, Rapid tumor shrinkage was seen bybatansine-0606 (15 mg/kg) from day 7. From day 10 onwards batansine-0606(15 mg/kg) treated tumors had shrunken to non-palpable. Compared to theunconjugated Bat0606, batansine-0606 more significantly inhibited thetumor growth as assessed by tumor weight measurements 49 days after drugtreatment.

Example 15 Preparation of Antibody-Drug Conjugates: D-Lmcc-Bat0606

Antibody Bat0606 was diluted to 2.5 mg/mL in solution A (50 mM potassiumphosphate, 50 mM NaCl, and 2 mM EDTA, pH 6.5). SMCC-MDC was added togive a ratio of SMCC-MDC to antibody of 7:1 mole equivalent. Then DMAwas added to 15% to the reaction and reaction was mixed by stirring for4 h at ambient temperature. D-Lmcc-bat0606 conjugate was purified fromexcess unreacted or hydrolyzed reagent and excess SMCC-MDC using a G25gel filtration column equilibrated in pH 7.4 phosphate buffer (aqueous).The conjugate was then dialyzed overnight into pH 7.4 phosphate buffer(aqueous) and then filtered through a 0.22 μm filter for final storage.The number of SMCC-MDC molecule per Abu molecule in the final conjugatewas measured by determining absorbance of the conjugate at 252 and 280nm and using known extinction coefficients for SMCC-MDC and antibody atthese two wavelengths. A ratio of maytansinoid compound to antibody of3.5:1.0 was normally obtained.

Example 16 Preparation of Antibody-Drug Conjugates: Batansine-0606

Antibody Bat0606 was diluted to 8.0 mg/mL in solution B (50 mM potassiumphosphate, 50 mM NaCl, and 2 mM EDTA, pH 8.0). Partial reduction wascarried out with (6 moles equivalent) DTT. After incubation at 37° C.for 60 minutes, the buffer was exchanged by elution through SephadexG-25 resin with solution B. The thiol-antibody value was determined fromthe reduced monoclonal antibody (mAb) concentration determined from280-nm absorbance, and the thiol concentration was determined byreaction with DTNB (5,5′-dithiobis(2-nitrobenzoic acid); Aldrich) andabsorbance measured at 412 nm.

The conjugation reaction was carried out with 10% DMA. The volume ofbatansine solution was calculated to contain 1.5-mol batansine (3AA-MDC)per mol equivalent of free thiol on the antibody. Batansine solution wasadded rapidly with mixing to the cold-reduced antibody solution, and themixture was stirred at r.t. for 3 hours, and continued for additional 1h after adding 5 mM cysteine. The reaction mixture was concentrated bycentrifugal ultrafiltration and buffer-exchanged by elution throughSephadex G25 equilibrated in PBS. The conjugate was then filteredthrough a 0.2-μm filter under sterile conditions and stored at −80° C.for analysis and testing. The Batansine-0606 was further analyzed fordrug/antibody ratio by measuring unreacted thiols with DTNB, and 3.5:1.0ratio of drug/antibody was often obtained. Batansine-0206 was furthercharacterized for concentration by UV absorbance, aggregation bysize-exclusion chromatography, and residual free drug by reverse-phaseHPLC. All mAbs and ADCs used in these studies exceeded 98% monomericprotein.

Example 17 Characterization of Anti-ErbB2 Antibody Drug ConjugateBatansine-0606

The growth inhibitory characteristics of Bat0606 and Batansine-0606 wereevaluated using the Her2 positive breast tumor cell line, SK-BR-3 (seeHudziak et al. Molec. Cell. Biol. 9(3):1165 1172 (1989) and Her2negative cell line A549 [Shanghai Cell Collections, Ltd., Co., Shanghai,China]. Briefly, cells were detached by using 0.25% (vol/vol) trypsinand suspended in complete medium. Aliquots of 100 μL containing 10,000cells for SK-BR-3 cell line and 8,000 cells for A549 cell line wereplated into 96-well microdilution plates. The cells were allowed toadhere overnight at 37° C., and 100 μL of media containing variousconcentrations of Bat0606 and Batansine-0606 was then added. After 72hours, plates were washed twice with PBS (pH 7.5), and analyzed forrelative cell proliferation with CCK-8 reagent. Drug conjugateBatansine-0606 more significantly inhibited the Her2 positive cellproliferation than naked Bat0606 (FIG. 20). Neither naked antibodyBat0606 nor drug conjugate Batansine-0606 inhibited the growth of Her2negative cell line A549 (FIG. 21).

Example 18 Characterization of Anti-ErbB2 Antibody Drug ConjugateD-Lmcc-Bat0606

The growth inhibitory characteristics of D-Lmcc-bat0606 were evaluatedusing the Her2 positive breast tumor cell line, SK-BR-3 (see Hudziak etal. Molec. Cell. Biol. 9(3):1165 1172 (1989) and Her2 negative cell lineA549. Briefly, cells were detached by using 0.25% (vol/vol) trypsin andsuspended in complete medium. Aliquots of 100 μL containing 10,000 cellsfor SK-BR-3 cell line and 8,000 cells for A549 cell line were platedinto 96-well microdilution plates. The cells were allowed to adhereovernight at 37° C., and 100 μL of media containing variousconcentrations of Bat0606 and D-Lmcc-Bat0606 was then added. After 72hours, plates were washed twice with PBS (pH 7.5), and analyzed forrelative cell proliferation with CCK-8 reagent. Drug conjugateD-Lmcc-Bat0606 strongly inhibited the Her2 positive cell proliferation(FIG. 22). Neither naked antibody Bat0606 nor drug conjugateD-Lmcc-Bat0606 inhibited the growth of Her2 negative cell line A549(FIG. 23).

Example 19

The stability studies of Batansine-0606 were evaluated in Sprague-Dawleyrats. Sprague-Dawley rats were administered 10 mg/kg Batansine-0606(based on the antibody component) by tail vein injection. Blood sampleswere collected from each mouse via the saphenous vein at 0 h, 10 min, 30min, 1 h, 2 h, 4 h, 8 h, 24 h, 36 h, day 2, day 3, day 4, day 7, day 14,day 21, day 28 after injection. Blood was collected into heparin coatedtubes followed by centrifugation (14,000×g, 3 minutes) to isolateplasma. Plasma concentrations of total Anti-Erb B/neu and antibody-drugconjugates were measured by ELISA. Total antibody concentration in theserum samples was measured as follows: 96-well ELISA plates were coatedwith HER2 ECD in 2 μg/mL carbonate/bicarbonate buffer (pH 9.6) at 4° C.overnight. After removal of the coat solution, nonspecific binding siteswere blocked by incubating with blocking solution (PBS, 1% BSA, 0.05%Tween 20) at room temperature for 1 hour. The plates were then washedwith wash buffer (0.05% Tween in PBS), and standards or samples dilutedin PBS were added. After a 2 h incubation, plates were washed and mouseanti-human IgG-horseradish peroxidase conjugate (Sigma, St. Louis, Mo.)was added for an additional 2 h. Plates were then washed again.Subsequently, 100 μL of 3,3,5,5-tetramethylbenzidine (Sigma, St. Louis,Mo.) were added to each well, and upon color development, the reactionwas stopped with 100 μL of 1 N sulfuric acid. Absorbance was measuredusing a VMax Kinetic Microplate reader (Molecular Devices, Sunnyvale,Calif.) at 490 nm. For measurement of antibody-drug conjugatesconcentration, wells were coated with HER2 ECD and serum samples addedas above. After the 2-h sample incubation, the plates were washed,rabbit anti-maytansine antibody was added to each well, and the plateswere incubated for 1 h. Plates were then washed, and HRP-conjugated goatanti-rabbit IgG (Sigma) was added for an additional 1 h incubation.Color detection and measurement were performed as described above.Noncompartmental pharmacokinetic parameters were calculated withWinNonlin (Pharsight, Mountain View, Calif.). The time-concentrationcurves of antibody component of Batansine-0606 and the drug component ofBatansine-0606 appeared to follow bi-exponential declines. The terminalhalf-life of antibody component of Batansine-0606 was 571.07, and theterminal half-life of drug component of Batansine-0606 was 88.36 hours.

Example 20 Batansine-0606 Eradicates Human NCI-N87 Tumor Xenografts

In Vivo Tumor Studies: The effects of batansine-0606(3AA-MDC-trastuzumab) on the growth of established tumors were examinedon human NCI-N87 tumor xenografts. Human NCI-N87 cells (ATCC, CRL-5822)were cultured in DMEM medium supplemented with 10% fetal bovine serum, 2mM glutamine and antibiotics. Female BALB/c nude mice, 4-8 weeks old,were injected subcutaneously with 5×10⁶ tumor cells in the dorsal areain a volume of 100 μL. When the tumor xenografts reaches a size of100-200 mm³ (calculated as 0.5× (length×width)), animals were thentreated with Bat0606 (5 or 15 mg/kg, i.v), batansine-0606 (5 or 15mg/kg, i.v), or control antibody (Rituximab, 15 mg/kg, i.v). Animalswere dosed once per week for a total of 5 doses i.v. in a volume of 100μL. Bat0606 treatment of NCI-N87 xenografts was discontinued on day 15and switched to batansine-0606 (5 or 15 mg/kg, i.v.) from the day 21onwards. Each group consisted of 10 mice. Tumor size was determined at 3days intervals. 42 days after tumor cell inoculation, animals wereeuthanized and tumors were removed and weighed. As shown in FIG. 29,rapid tumor shrinkage was seen by batansine-0606 (5 or 15 mg/kg) fromday 11. From day 32 onwards batansine-0606 (5 or 15 mg/kg) treatedtumors had shrunken to non-palpable. Compared to the unconjugatedBat0606, at 5 or 15 mg/kg dose tested, batansine-0606 more significantlyinhibited the tumor growth as assessed by tumor weight measurements 35days after drug treatment.

Example 21 Preparation of Antibody-Drug Conjugates: D-Lmcc-Bat1206

Antibody Bat1206 was diluted to 2.5 mg/mL in solution A (50 mM potassiumphosphate, 50 mM NaCl, and 2 mM EDTA, pH 6.5). SMCC-MDC was added togive a ratio of SMCC-MDC to antibody of 7:1 mole equivalent. Then DMAwas added to 15% to the reaction and reaction was mixed by stirring for4 h at ambient temperature. D-Lmcc-Bat1206 conjugate was purified fromexcess unreacted or hydrolyzed reagent and excess SMCC-MDC using a G25gel filtration column equilibrated in pH 7.4 phosphate buffer (aqueous).The conjugate was then dialyzed overnight into pH 7.4 phosphate buffer(aqueous) and then filtered through a 0.22 μm filter for final storage.The number of SMCC-MDC molecule per Abu molecule in the final conjugatewas measured by determining absorbance of the conjugate at 252 and 280nm and using known extinction coefficients for SMCC-MDC and antibody atthese two wavelengths. A ratio of maytansinoid compound to antibody of2-5 to 1 was normally obtained.

Example 22 Preparation of Anti-CD20 Antibody Drug ConjugateBatansine-1206

Antibody Bat1206 was diluted to 8.0 mg/mL in solution B (50 mM potassiumphosphate, 50 mM NaCl, and 2 mM EDTA, pH 8.0). Partial reduction wascarried out with (6 moles equivalent) DTT. After incubation at 37° C.for 60 minutes, the buffer was exchanged by elution through SephadexG-25 resin with solution B. The thiol-antibody value was determined fromthe reduced monoclonal antibody (mAb) concentration determined from280-nm absorbance, and the thiol concentration was determined byreaction with DTNB (5,5′-dithiobis(2-nitrobenzoic acid); Aldrich) andabsorbance measured at 412 nm. The conjugation reaction was carried outwith 10% DMA. The volume of batansine solution was calculated to contain1.5-mol batansine (3AA-MDC) per mol equivalent of free thiol on theantibody. Batansine solution was added rapidly with mixing to thecold-reduced antibody solution, and the mixture was stirred at r.t. for3 hours, and continued for additional 1 h after adding 5 mM cysteine.The reaction mixture was concentrated by centrifugal ultrafiltration andbuffer-exchanged by elution through Sephadex G25 equilibrated in PBS.The conjugate was then filtered through a 0.2-μm filter under sterileconditions and stored at −80° C. for analysis and testing. TheBatansine-1206 was further analyzed for drug/antibody ratio by measuringunreacted thiols with DTNB, and 3.5:1 ratio of drug/antibody was oftenobtained. Batansine-1206 was further characterized for concentration byUV absorbance, aggregation by size-exclusion chromatography, andresidual free drug by reverse-phase HPLC.

Example 23 Study on the Effect of Bat1206 and D-Lmcc-Bat1206 on theGrowth of Raji Cells

The effect of Bat1206 and D-Lmcc-Bat1206 on the growth of Raji cell(Shanghai Cell Collection. Ltd., Co. Shanghai, China) was determinedusing the method described by Ishiyama et al. (Biol. Pharmacol. Bull.,19: 1518-1520, 1996). Briefly, 10 thousands cells in 100 μL of DMEM:F12(GBICO, CA) culture medium without serum were seeded into each well of a96-well plate. Twice-fold serial dilutions of antibodies or conjugatedform were prepared and each diluted antibodies were added in triplicateto the wells and the cultures were incubated at 37° C. for 3 days. Thecontrols consisted of either medium alone or medium containing of Rajicell. After incubation, CCK-8 was added to each well and the absorbanceat 450 nm of each well was determined in a Spectra Max spectrophotometer(Molecular Devices, Sunnyvale, Calif.). As shown in FIG. 26,D-Lmcc-Bat1206 more effectively inhibited CD20 positive cell growth thannon conjugated anti-CD20 antibody Bat1206.

Example 24 Cellular Metabolites of Batansine-0206 and D-Lmcc-Bat0206

Cellular metabolites of Batansine-0206 and D-Lmcc-Bat0206 were assayedas described in Erickson, et al. Cancer Res 66:4426-4433 (2006).Briefly, A431 cells (6×10⁶) suspended in 3 mL culture medium containingBatansine-0206 at a concentration of 10-7 mol/L of conjugated antibodywere incubated at 37° C. for 3 to 30 hours. The cells and the mediumwere then separated by centrifugation (2,000 g, 5 minutes). Thesupernatant (3 mL) was chilled on ice, mixed with 4 mL ice-cold acetone,and kept at −80° C. for at least 1 hour or until further processing.Precipitated protein was removed by centrifugation at 2,500 g and thesupernatants were acidified with 5% acetic acid and evaporated todryness. The samples were dissolved in 0.12 mL of 20% aqueous CH₃CNcontaining 0.025% trifluoroacetic acid (TFA), aliquots of 0.1 mL weresubmitted to LC-MS. The metabolite of Batansine-0206: LC-MS (M+H⁺)calc.: 964.5. found: 964.2. The metabolite of D-Lmcc-Bat0206: LC-MS(M+H⁺) calc.: 1103.7. found: 1103.3.

1.-57. (canceled)
 58. A compound of Formula I or I-1:

or a salt thereof, wherein X is hydrogen or halo; Y is selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and—C(═O)R⁵; R¹ is selected from the group consisting of hydrogen, —OH,—OC(═O)R⁵ and —OR⁵; R² is hydrogen or C₁-C₆ alkyl; R³ is methyl, —CH₂OH,or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵ is C₁-C₆ alkyl or benzyl; R⁶ isC₁-C₆ alkyl, phenyl or benzyl; R⁷ is hydrogen, C₁-C₆ alkyl or an aminoacid side chain; R⁸ is hydrogen or C₁-C₆ alkyl; each Z is independentlyhydrogen or C₁-C₄ alkyl, or the two Z with the carbon atom to which theyare attached form a C═O; and in Formula I, L is selected fromsubstituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene substituted C₁-C₂₀alkylene wherein one or more of the —CH₂— groups are independentlyreplaced with C₃-C₈ cycloalkylene, —S—, —NR⁸C(═O)—, —SO₂NR⁸—, or—NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substitutedwith 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is independentlyC₁₋₆ alkyl substituted with 1 or 2 substituents independently selectedfrom the group consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and—NR¹¹R¹¹, wherein each R¹¹ is independently hydrogen, alkyl, aryl,cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹together with the nitrogen form a heterocyclic, wherein the heterocyclicis optionally substituted with one or two oxo; in Formula I-1, L isselected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein one ormore of the —CH₂— groups are independently replaced with C₃-C₈cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(═O)NR⁸—, —NR⁸C(═O)—,—SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylenesubstituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ isindependently C₁₋₆ alkyl optionally substituted with 1 or 2 substituentsindependently selected from the group consisting of —SH, —S—C₁₋₄ alkyl,—CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ is independentlyhydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic, or the two R¹¹ together with the nitrogen form aheterocyclic, wherein the heterocyclic is optionally substituted withone or two oxo.
 59. The compound of claim 58, which is:

or a salt thereof.
 60. The compound of claim 58, which is a compound ofFormula III or III-1:

or a salt thereof, wherein X is H or Cl; Y is H or methyl; R⁷ ishydrogen, C₁-C₆ alkyl or an amino acid side chain; R⁸ is hydrogen orC₁-C₆ alkyl; and in Formula III, L is selected from substituted C₁-C₂₀alkylene, C₃-C₈ cycloalkylene substituted C₁-C₂₀ alkylene wherein one ormore of the —CH₂— groups are independently replaced with C₃-C₈cycloalkylene, —S—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substitutedC₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with 1 to 4 —SO₃H,—P(O)(OH)₂ or R²³, wherein each R²³ is independently C₁₋₆ alkylsubstituted with 1 or 2 substituents independently selected from thegroup consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹,wherein each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹ together withthe nitrogen form a heterocyclic, wherein the heterocyclic is optionallysubstituted with one or two oxo; in Formula III-1, L is selected fromoptionally substituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, optionallysubstituted C₁-C₂₀ alkylene wherein one or more of the —CH₂— groups areindependently replaced with C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—,—C(O)—, —C(═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substitutedC₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with 1 to 4 —SO₃H,—P(O)(OH)₂ or R²³, wherein each R²³ is independently C₁₋₆ alkyloptionally substituted with 1 or 2 substituents independently selectedfrom the group consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and—NR¹¹R¹¹, wherein each R¹¹ is independently hydrogen, alkyl, aryl,cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹together with the nitrogen form a heterocyclic, wherein the heterocyclicis optionally substituted with one or two oxo.
 61. The compound of claim58, which is a compound of Formula IV-1:

or a salt thereof, wherein X is H or Cl; Y is H or methyl; R⁷ ishydrogen, C₁-C₆ alkyl or an amino acid side chain; R⁸ is hydrogen orC₁-C₆ alkyl; and m is an integer of 1 to
 20. 62. The compound of claim58, selected from the group consisting of

or a salt thereof.
 63. The compound of claim 58, selected from the groupconsisting of

or a salt thereof.
 64. The compound of claim 58, which is a compound ofFormula V-1:

or a salt thereof.
 65. A compound of Formula II or II-1:

or a salt thereof, wherein X is hydrogen or halo; Y is selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and—C(C═O)R⁵; R¹ is selected from the group consisting of hydrogen, —OH,—OC(═O)R⁵ and —OR⁵; R² is hydrogen or C₁-C₆ alkyl; R³ is methyl, —CH₂OH,or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵ is C₁-C₆ alkyl or benzyl; R⁶ isC₁-C₆ alkyl, phenyl or benzyl; R⁷ is hydrogen, C₁-C₆ alkyl or an aminoacid side chain; R⁸ is hydrogen or C₁₋₆ alkyl; each Z is independentlyhydrogen or C₁-C₄ alkyl, or the two Z with the carbon atom to which theyare attached form a C═O; and L is selected from optionally substitutedC₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, optionally substituted C₁-C₂₀alkylene wherein one or more of the —CH₂— group is independentlyreplaced with C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—,—NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀alkylene substituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein eachR²³ is independently C₁₋₆ alkyl optionally substituted with 1 or 2substituents independently selected from the group consisting of —SH,—S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ isindependently hydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl,heteroaryl, and heterocyclic, or the two R¹¹ together with the nitrogenform a heterocyclic, wherein the heterocyclic is optionally substitutedwith one or two oxo.
 66. The compound of claim 65, which is:

or a salt thereof.
 67. A compound of Formula Ia or Ia-1:

or a pharmaceutically acceptable salt or solvate thereof, wherein X ishydrogen or halo; Y is selected from the group consisting of hydrogen,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵; R¹ is selected from thegroup consisting of hydrogen, —OH, —OC(═O)R⁵ and —OR⁵; R² is hydrogen orC₁-C₆ alkyl; R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵is C₁-C₆ alkyl or benzyl; R⁶ is C₁-C₆ alkyl, phenyl or benzyl; R⁷ ishydrogen, C₁-C₆ alkyl or an amino acid side chain; R⁸ is hydrogen orC₁₋₆ alkyl; p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; inFormula Ia, L is selected from substituted C₁-C₂₀ alkylene, C₃-C₈cycloalkylene, substituted C₁-C₂₀ alkylene wherein one or more of the—CH₂— groups are independently replaced with C₃-C₈ cycloalkylene, —S—,—NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀alkylene substituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein eachR²³ is independently C₁₋₆ alkyl substituted with 1 or 2 substituentsindependently selected from the group consisting of —SH, —S—C₁₋₄ alkyl,—CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ is independentlyhydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic, or the two R¹¹ together with the nitrogen form aheterocyclic, wherein the heterocyclic is optionally substituted withone or two oxo; in Formula Ia-1, L is selected from optionallysubstituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, optionally substitutedC₁-C₂₀ alkylene wherein one or more of the —CH₂— groups areindependently replaced with C₃-C₈ cycloalkylene, —O—, —S—, —C(O)—,—C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀alkylene is C₁-C₂₀ alkylene substituted with 1 to 4 —SO₃H, —P(O)(OH)₂ orR²³, wherein each R²³ is independently C₁₋₆ alkyl optionally substitutedwith 1 or 2 substituents independently selected from the groupconsisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹,wherein each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹ together withthe nitrogen form a heterocyclic, wherein the heterocyclic is optionallysubstituted with one or two oxo; and Abu is an antigen binding unit. 68.The compound of claim 67 which is:

or a salt thereof.
 69. The compound of claim 67, which is of FormulaIIIa or IIIa-1:

or a pharmaceutically acceptable salt or solvate thereof, wherein X is Hor Cl; Y is H or methyl; R⁷ is hydrogen, C₁-C₆ alkyl or an amino acidside chain; R⁸ is hydrogen or C₁-C₆ alkyl; p is selected from 1, 2, 3,4, 5, 6, 7, 8, 9, and 10; in Formula IIIa, L is selected fromsubstituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, substituted C₁-C₂₀alkylene wherein one or more of the —CH₂— groups are independentlyreplaced with C₃-C₈ cycloalkylene, —S—, —NR⁸C(═O)—, —SO₂NR⁸—, or—NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substitutedwith 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is independentlyC₁₋₆ alkyl optionally substituted with 1 or 2 substituents independentlyselected from the group consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹,—CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ is independently hydrogen, alkyl,aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic, or the twoR¹¹ together with the nitrogen form a heterocyclic, wherein theheterocyclic is optionally substituted with one or two oxo; in FormulaIIIa-1, L is selected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein one ormore of the —CH₂— groups are independently replaced with C₃-C₈cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—, —NR⁸C(═O)—,—SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylenesubstituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ isindependently C₁₋₆ alkyl optionally substituted with 1 or 2 substituentsindependently selected from the group consisting of —SH, —S—C₁₋₄ alkyl,—CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ is independentlyhydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic, or the two R¹¹ together with the nitrogen form aheterocyclic, wherein the heterocyclic is optionally substituted withone or two oxo; and Abu is an antigen binding unit.
 70. The compound ofclaim 67, which is of Formula IVa-1:

or a pharmaceutically acceptable salt or solvate thereof, wherein X is Hor Cl; Y is H or methyl; R⁷ is hydrogen, C₁-C₆ alkyl or an amino acidside chain; R⁸ is hydrogen or C₁-C₆ alkyl; p is selected from 1, 2, 3,4, 5, 6, 7, 8, 9, and 10; m is selected from an integer of 1 to 20; andAbu is an antigen binding unit.
 71. The compound of claim 67, selectedfrom the group consisting of

or a pharmaceutically acceptable salt thereof.
 72. The compound of claim67, selected from the group consisting of

or a pharmaceutically acceptable salt thereof.
 73. A compound of FormulaIIa or IIa-1:

or a pharmaceutically acceptable salt or solvate thereof, wherein X ishydrogen or halo; Y is selected from the group consisting of hydrogen,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵; R¹ is selected from thegroup consisting of hydrogen, —OH, —OC(═O)R⁵ and —OR⁵; R² is hydrogen orC₁-C₆ alkyl; R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵is C₁-C₆ alkyl or benzyl; R⁶ is C₁-C₆ alkyl, phenyl or benzyl; R⁷ ishydrogen, C₁-C₆ alkyl or an amino acid side chain; R⁸ is hydrogen orC₁₋₆ alkyl; p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each Zis independently hydrogen or C₁-C₄ alkyl, or the two Z with the carbonatom to which they are attached form a C═O; in Formula IIa, L isselected from substituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene,substituted C₁-C₂₀ alkylene wherein one or more of the —CH₂— groups areindependently replaced with C₃-C₈ cycloalkylene, —S—, —NR⁸C(═O)—,—SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylenesubstituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ isindependently C₁₋₆ alkyl substituted with 1 or 2 substituentsindependently selected from the group consisting of —SH, —S—C₁₋₄ alkyl,—CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ is independentlyhydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic, or the two R¹¹ together with the nitrogen form aheterocyclic, wherein the heterocyclic is optionally substituted withone or two oxo; in Formula IIa-1, L is selected from optionallysubstituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, optionally substitutedC₁-C₂₀ alkylene wherein one or more of the —CH₂— groups areindependently replaced with C₃-C₈ cycloalkylene, —O—, —S—, —NR⁸—,—C(O)—, —C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substitutedC₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with 1 to 4 —SO₃H,—P(O)(OH)₂ or R²³, wherein each R²³ is independently C₁₋₆ alkyloptionally substituted with 1 or 2 substituents independently selectedfrom the group consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and—NR¹¹R¹¹, wherein each R¹¹ is independently hydrogen, alkyl, aryl,cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹together with the nitrogen form a heterocyclic, wherein the heterocyclicis optionally substituted with one or two oxo; and Abu is an antigenbinding unit.
 74. The compound of claim 73, which is:

or a pharmaceutically acceptable salt or solvate thereof.
 75. Thecompound of claim 67, wherein Abu is an antigen binding unit withbinding specificity to human EGFR.
 76. The compound of claim 67, whereinAbu is an antigen binding unit with binding specificity to human CD20.77. The compound of claim 67, wherein Abu is an antigen binding unitwith binding specificity to human Her2.
 78. The compound of claim 75,wherein Abu is an antibody, antibody fragment, or cell specific ligands.79. The compound of claim 67, wherein Abu is an antibody comprising SEQID 1 and 2, or SEQ ID 3 and 4, or SEQ ID 5 and 6, or SEQ ID 7 and 8, orSEQ ID 9 and 10, SEQ ID 11 and 12, or SEQ ID 13 and
 14. 80. The compoundof claim 67, wherein Abu is an antibody comprising Bat0202 (SEQ IDNO: 1) and/or Bat0204 (SEQ ID NO: 2).
 81. The compound of claim 67,wherein Abu is Bat0206.
 82. The compound of claim 67, wherein Abu isselected from C225, EGF-ABX, EGF-ABX, NIMO, Matu, rituxamab, Cetuximab,trastuzumab and Pertuzumab.
 83. A drug-linker-antigen binding unitconjugate wherein the drug is a maytansinoid and the antigen bindingunit is Bat0206.
 84. A compound of Formula Id or IId:

or a pharmaceutically acceptable salt or solvate thereof, wherein X ishydrogen or halo; Y is selected from the group consisting of hydrogen,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵; R¹ is selected from thegroup consisting of hydrogen, —OH, —OC(═O)R⁵ and —OR⁵; R² is hydrogen orC₁-C₆ alkyl; R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; andR⁵ is C₁-C₆ alkyl or benzyl; R⁶ is C₁-C₆ alkyl, phenyl or benzyl; R⁷ ishydrogen, C₁-C₆ alkyl or an amino acid side chain; R⁸ is hydrogen orC₁₋₆ alkyl; p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and inFormula Id, L^(b) is selected from C₁-C₂₀ alkylene optionallysubstituted with C₁-C₄ alkyl, —SO₃H or —P(O)(OH)₂, C₃-C₈ cycloalkyleneoptionally substituted with C₁-C₄ alkyl, and C₁-C₂₀ alkylene which isoptionally substituted with C₁-C₄ alkyl, —SO₃H or —P(O)(OH)₂ and whereinone or more of the —CH₂— groups are independently replaced with C₃-C₈cycloalkylene, —S—, —S—S—, —SO₂NR⁸—, or —NR⁶SO₂; in Formula IId, L^(b)is selected from C₁-C₂₀ alkylene optionally substituted with C₁-C₄alkyl, —SO₃H or —P(O)(OH)₂, C₃-C₈ cycloalkylene optionally substitutedwith C₁-C₄ alkyl, and C₁-C₂₀ alkylene which is optionally substitutedwith C₁-C₄ alkyl, —SO₃H or —P(O)(OH)₂ and wherein one or more of the—CH₂— groups are independently replaced with C₃-C₈ cycloalkylene, 3-8membered heterocycloalkylene optionally substituted with one or moreC₁-C₄ alkyl or oxo, —O—, —S—, —S—S—, —NR⁸—, —C(C═O)NR⁸—, —NR⁸C(═O)—,—SO₂NR⁸—, or —NR⁸SO₂; BAT0206 is an Anti-EGFR antibody.
 85. The compoundof claim 84, wherein, in Formula Id, L^(b) is C₁-C₂₀ alkylene which issubstituted with C₁-C₄ alkyl, —SO₃H or —P(O)(OH)₂ and wherein one ormore of the —CH₂— groups are independently replaced with —S—, —S—S—,


86. The compound of claim 84, selected from the group consisting of

or a pharmaceutically acceptable salt or solvate thereof, wherein X ishydrogen or halo; Y is selected from the group consisting of hydrogen,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and —C(C═O)R⁵; R¹ is selected from thegroup consisting of hydrogen, —OH, —OC(═O)R⁵ and —OR⁵; R² is hydrogen orC₁-C₆ alkyl; R³ is methyl, —CH₂OH, or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵is C₁-C₆ alkyl or benzyl; R⁶ is C₁-C₆ alkyl, phenyl or benzyl R⁷ ishydrogen, C₁-C₆ alkyl or an amino acid side chain; R⁸ is hydrogen orC₁₋₆ alkyl; p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and Lis selected from substituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylenesubstituted C₁-C₂₀ alkylene wherein one or more of the —CH₂— groups areindependently replaced with C₃-C₈ cycloalkylene, —S—, —NR⁸C(═O)—,—SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylenesubstituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ isindependently C₁₋₆ alkyl substituted with 1 or 2 substituentsindependently selected from the group consisting of —SH, —S—C₁₋₄ alkyl,—CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ is independentlyhydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic, or the two R¹¹ together with the nitrogen form aheterocyclic, wherein the heterocyclic is optionally substituted withone or two oxo; and BAT0206 is an Anti-EGFR antibody.
 87. The compoundof claim 84, selected from the group consisting of

or a pharmaceutically acceptable salt or solvate thereof, wherein X is Hor Cl; Y is H or methyl; R⁷ is hydrogen, C₁-C₆ alkyl or an amino acidside chain; R⁸ is hydrogen or C₁-C₆ alkyl; p is selected from 1, 2, 3,4, 5, 6, 7, 8, 9, and 10; and BAT0206 is an Anti-EGFR antibody.
 88. Thecompound of claim 84, selected from the group consisting of

or a pharmaceutically acceptable salt or solvate thereof, whereinBAT0206 is an Anti-EGFR antibody.
 89. A compound of Formula Ib or Ib-1:

or a salt thereof, wherein X is hydrogen or halo; Y is selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and—C(C═O)R⁵; R¹ is selected from the group consisting of hydrogen, —OH,—OC(═O)R⁵ and —OR⁵; R² is hydrogen or C₁-C₆ alkyl; R³ is methyl, —CH₂OH,or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵ is C₁-C₆ alkyl or benzyl; R⁶ isC₁-C₆ alkyl, phenyl or benzyl; R⁷ is hydrogen, C₁-C₆ alkyl or an aminoacid side chain; R⁸ is hydrogen or C₁₋₆ alkyl; each Z is independentlyhydrogen or C₁-C₄ alkyl, or the two Z with the carbon atom to which theyare attached form a C═O; in Formula Ib, L is selected from substitutedC₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, substituted C₁-C₂₀ alkylenewherein one or more of the —CH₂— groups are independently replaced withC₃-C₈ cycloalkylene, —S—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substitutedC₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with 1 to 4 —SO₃H,—P(O)(OH)₂ or R²³, wherein each R²³ is independently C₁₋₆ alkylsubstituted with 1 or 2 substituents independently selected from thegroup consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹,wherein each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹ together withthe nitrogen form a heterocyclic, wherein the heterocyclic is optionallysubstituted with one or two oxo; in Formula Ib-1, L is selected fromoptionally substituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, optionallysubstituted C₁-C₂₀ alkylene wherein one or more of the —CH₂— groups areindependently replaced with C₃-C₈ cycloalkylene, —O—, —S—, —C(O)—,—C(C═O)NR⁸—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀alkylene is C₁-C₂₀ alkylene substituted with 1 to 4 —SO₃H, —P(O)(OH)₂ orR²³, wherein each R²³ is independently C₁₋₂₀ alkyl optionallysubstituted with 1 or 2 substituents independently selected from thegroup consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹,wherein each R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹ together withthe nitrogen form a heterocyclic, wherein the heterocyclic is optionallysubstituted with one or two oxo; and AA is an amino acid or thiolatedamino acid.
 90. The compound of claim 87, which is selected from thegroup consisting of

or a salt thereof, wherein X is H or Cl; Y is H or methyl; R⁷ ishydrogen, C₁-C₆ alkyl or an amino acid side chain; R⁸ is hydrogen orC₁-C₆ alkyl; in Formulae IIIb′ and IIIb, L is selected from substitutedC₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, substituted C₁-C₂₀ alkylenewherein one or more of the —CH₂— groups are independently replaced withC₃-C₈ cycloalkylene, —S—, —NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substitutedC₁-C₂₀ alkylene is C₁-C₂₀ alkylene substituted with 1 to 4 —SO₃H,—P(O)(OH)₂ or R²³, wherein each R²³ is independently C₁₋₈ alkylsubstituted with 1 or 2 substituents independently selected from thegroup consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and whereineach R¹¹ is independently hydrogen, alkyl, aryl, cycloalkyl,cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹ together withthe nitrogen form a heterocyclic, wherein the heterocyclic is optionallysubstituted with one or two oxo; in Formulae IIIb′-1 and IIIb-1, L isselected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein one ormore of the —CH₂— groups are independently replaced with C₃-C₈cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—, —NR⁸C(═O)—,—SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylenesubstituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ isindependently C₁₋₆ alkyl optionally substituted with 1 or 2 substituentsindependently selected from the group consisting of —SH, —S—C₁₋₄ alkyl,—CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ is independentlyhydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic, or the two R¹¹ together with the nitrogen form aheterocyclic, wherein the heterocyclic is optionally substituted withone or two oxo; and AA is an amino acid or thiolated amino acid.
 91. Thecompound of claim 89, which is of Formula IVb-1:

or a salt thereof, wherein X is H or Cl; Y is H or methyl; R⁷ ishydrogen, C₁-C₆ alkyl or an amino acid side chain; R⁸ is hydrogen orC₁-C₆ alkyl; m is selected from an integer of 1 to 20; and AA is anamino acid or thiolated amino acid.
 92. The compound of claim 89,wherein AA is

wherein

represents point of connection to the rest of the molecule.
 93. Thecompound of claim 89, selected from the group consisting of

or a salt thereof, wherein X is hydrogen or halo; Y is selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and—C(C═O)R⁵; R¹ is selected from the group consisting of hydrogen, —OH,—OC(═O)R⁵ and —OR⁵; R² is hydrogen or C₁-C₆ alkyl; R³ is methyl, —CH₂OH,or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵ is C₁-C₆ alkyl or benzyl; R⁶ isC₁-C₆ alkyl, phenyl or benzyl R⁷ is hydrogen, C₁-C₆ alkyl or an aminoacid side chain; R⁸ is hydrogen or C₁₋₆ alkyl; L is selected fromsubstituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, substituted C₁-C₂₀alkylene wherein one or more of the —CH₂— groups are independentlyreplaced with C₃-C₈ cycloalkylene, —S—, —NR⁸C(═O)—, —SO₂NR⁸—, or—NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substitutedwith 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is independentlyC₁₋₆ alkyl substituted with 1 or 2 substituents independently selectedfrom the group consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and—NR¹¹R¹¹, wherein each R¹¹ is independently hydrogen, alkyl, aryl,cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹together with the nitrogen form a heterocyclic, wherein the heterocyclicis optionally substituted with one or two oxo; and m is selected from aninteger of 1 to
 20. 94. A compound selected from the group consistingof:

or a salt thereof, wherein X is hydrogen or halo; Y is selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and—C(C═O)R⁵; R¹ is selected from the group consisting of hydrogen, —OH,—OC(═O)R⁵ and —OR⁵; R² is hydrogen or C₁-C₆ alkyl; R³ is methyl, —CH₂OH,or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵ is C₁-C₆ alkyl or benzyl; R⁶ isC₁-C₆ alkyl, phenyl or benzyl; R⁷ is hydrogen, C₁-C₆ alkyl or an aminoacid side chain; R⁸ is hydrogen or C₁₋₆ alkyl; each Z is independentlyhydrogen or C₁-C₄ alkyl, or the two Z with the carbon atom to which theyare attached form a C═O; L is selected from optionally substitutedC₁-C₂₀ alkylene, C₃-C₅ cycloalkylene, optionally substituted C₁-C₂₀alkylene wherein one or more of the —CH₂— groups are independentlyreplaced with C₃-C₈ cycloalkylene, —O—, —S—, —NR⁶—, —C(O)—, —C(═O)NR⁸—,—NR⁸C(═O)—, —SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀alkylene substituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein eachR²³ is independently C₁₋₆ alkyl optionally substituted with 1 or 2substituents independently selected from the group consisting of —SH,—S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and wherein each R¹¹ is independentlyhydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic, or the two R¹¹ together with the nitrogen form aheterocyclic, wherein the heterocyclic is optionally substituted withone or two oxo; and AA is an amino acid or thiolated amino acid.
 95. Apharmaceutical composition comprising a compound of claim
 67. 96. Amethod of treating a proliferative, inflammatory or immunologic diseaseor condition in a patient in need thereof comprising administering aneffective amount of a compound of claim
 67. 97. A method of treating aproliferative, inflammatory or immunologic disease or condition in apatient in need thereof comprising administering an effective amount ofa compound of Formula Ib or Ib-1,

wherein the compound of Formula Ib or Ib-1 is generated as a result of ametabolic chemical reaction following administration of a compound ofclaim 66 to the patient. wherein X is hydrogen or halo; Y is selectedfrom the group consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl,and —C(C═O)R⁵; R¹ is selected from the group consisting of hydrogen,—OH, —OC(═O)R⁵ and —OR⁵; R² is hydrogen or C₁-C₆ alkyl; R³ is methyl,—CH₂OH, or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵ is C₁-C₆ alkyl or benzyl;R⁶ is C₁-C₆ alkyl, phenyl or benzyl; R⁷ is hydrogen, C₁-C₆ alkyl or anamino acid side chain; R⁶ is hydrogen or C₁₋₆ alkyl; each Z isindependently hydrogen or C₁-C₄ alkyl, or the two Z with the carbon atomto which they are attached form a C═O; in Formula Ib, L is selected fromsubstituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, substituted C₁-C₂₀alkylene wherein one or more of the —CH₂— groups are independentlyreplaced with C₃-C₈ cycloalkylene, —S—, —NR⁸C(═O)—, —SO₂NR⁸—, or—NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substitutedwith 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is independentlyC₁₋₆ alkyl substituted with 1 or 2 substituents independently selectedfrom the group consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and—NR¹¹R¹¹, wherein each R¹¹ is independently hydrogen, alkyl, aryl,cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹together with the nitrogen form a heterocyclic, wherein the heterocyclicis optionally substituted with one or two oxo; in Formula Ib-1, L isselected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein one ormore of the —CH₂— groups are independently replaced with C₃-C₈cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—, —NR⁸C(═O)—,—SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylenesubstituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ isindependently C₁₋₆ alkyl optionally substituted with 1 or 2 substituentsindependently selected from the group consisting of —SH, —S—C₁₋₄ alkyl,—CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ is independentlyhydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic, or the two R¹¹ together with the nitrogen form aheterocyclic, wherein the heterocyclic is optionally substituted withone or two oxo; and AA is an amino acid or thiolated amino acid.
 98. Themethod of claim 97, wherein the compound of Formula Ib or Ib-1 is


99. A method of treating a proliferative, inflammatory or immunologicdisease or condition in a patient in need thereof comprisingadministering an effective amount of a compound of Formula IIb or IIb-1,

wherein the compound of Formula IIb or IIb-1 is generated as a result ofa metabolic chemical reaction following administration of a compound ofclaim 71 to the patient.
 100. A method of treating a proliferative,inflammatory or immunologic disease or condition in a patient in needthereof comprising administering an effective amount of a compound ofFormula Vc,

wherein the compound of Formula Vc is generated as a result of ametabolic chemical reaction following administration of a compound ofFormula VIa or VIIa,

or a pharmaceutically acceptable salt thereof, to the patient, whereinAbu described in Formula VIa and VIIa is antigen binding unit; and p isselected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and
 10. 101. A method oftreating a proliferative, inflammatory or immunologic disease orcondition in a patient in need thereof comprising administering aneffective amount of a compound of Formula Vc,

wherein the compound of Formula Vc is generated as a result of ametabolic chemical reaction following administration of a compound ofFormula XVa, XVIa, XVIIIa, or XIXa

or a pharmaceutically acceptable salt thereof, to the patient, whereinAbu described in Formula XVa, XVIa, XVIIIa, or XIXa is antigen bindingunit; and p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and
 10. 102. Amethod of treating a proliferative, inflammatory or immunologic diseaseor condition in a patient in need thereof comprising administering aneffective amount of a compound of Formula VIIIc,

wherein the compound of Formula VIIIc is generated as a result of ametabolic chemical reaction following administration of a compound ofFormula VIIIa,

or a pharmaceutically acceptable salt thereof, to the patient, whereinAbu is antigen binding unit; p is selected from 1, 2, 3, 4, 5, 6, 7, 8,9, and
 10. 103. A method of treating a proliferative, inflammatory orimmunologic disease or condition in a patient in need thereof comprisingadministering an effective amount of a compound of Formula IXc,

wherein the compound of Formula IXc is generated as a result of ametabolic chemical reaction following administration of a compound ofFormula IXa,

or a pharmaceutically acceptable salt thereof, to the patient whereinAbu is antigen binding unit; p is selected from 1, 2, 3, 4, 5, 6, 7, 8,9, and
 10. 104. A method of treating a proliferative, inflammatory orimmunologic disease or condition in a patient in need thereof comprisingadministering an effective amount of a compound of Formula Xc,

wherein the compound of Formula Xc is generated as a result of ametabolic chemical reaction following administration of a compound ofFormula Xa,

or a pharmaceutically acceptable salt thereof, to the patient, whereinAbu is antigen binding unit; p is selected from 1, 2, 3, 4, 5, 6, 7, 8,9, and
 10. 105. A method of treating a proliferative, inflammatory orimmunologic disease or condition in a patient in need thereof comprisingadministering an effective amount of a compound of Formula XIc,

wherein the compound of Formula XIc is generated as a result of ametabolic chemical reaction following administration of a compound ofFormula XIa,

or a pharmaceutically acceptable salt thereof, to the patient. whereinAbu is antigen binding unit; p is selected from 1, 2, 3, 4, 5, 6, 7, 8,9, and
 10. 106. A method of treating a proliferative, inflammatory orimmunologic disease or condition in a patient in need thereof comprisingadministering an effective amount of a compound of Formula XIIc,

wherein the compound of Formula XIIc is generated as a result of ametabolic chemical reaction following administration of a compound ofFormula XIIa,

or a pharmaceutically acceptable salt thereof, to the patient, whereinAbu is antigen binding unit; p is selected from 1, 2, 3, 4, 5, 6, 7, 8,9, and
 10. 107. A method of treating a proliferative, inflammatory orimmunologic disease or condition in a patient in need thereof comprisingadministering an effective amount of a compound of Formula XIVc,

wherein the compound of Formula XIVc is generated as a result of ametabolic chemical reaction following administration of a compound ofFormula XIVa,

or a pharmaceutically acceptable salt thereof, to the patient. whereinAbu is antigen binding unit; p is selected from 1, 2, 3, 4, 5, 6, 7, 8,9, and
 10. 108. A method of treating a proliferative, inflammatory orimmunologic disease or condition in a patient in need thereof comprisingadministering an effective amount of a compound of Formula XVIIc,

wherein the compound of Formula XVIIc is generated as a result of ametabolic chemical reaction following administration of a compound ofFormula XVIIa,

or a pharmaceutically acceptable salt thereof, to the patient. whereinAbu is antigen binding unit; p is selected from 1, 2, 3, 4, 5, 6, 7, 8,9, and
 10. 109. The method of claim 97, wherein the antigen binding unitis an antibody comprising SEQ ID 1 and 2, or SEQ ID 3 and 4, or SEQ ID 5and 6, or SEQ ID 7 and 8, or SEQ ID 9 and 10, SEQ ID 11 and 12, or SEQID 13 and 14, or an equivalent thereof.
 110. A method of preparing acompound of claim 67 or a pharmaceutically acceptable salt thereof,comprising contacting an antigen binding unit with a compound of FormulaI or I-1:

or a salt thereof, wherein X is hydrogen or halo; Y is selected from thegroup consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and—C(C═O)R⁵; R¹ is selected from the group consisting of hydrogen, —OH,—OC(═O)R⁵ and —OR⁵; R² is hydrogen or C₁-C₆ alkyl; R³ is methyl, —CH₂OH,or —CH₂C(═O)R⁶; R⁴ is —OH or —SH; R⁵ is C₁-C₆ alkyl or benzyl; R⁶ isC₁-C₆ alkyl, phenyl or benzyl; R⁷ is hydrogen, C₁-C₆ alkyl or an aminoacid side chain; R⁸ is hydrogen or C₁₋₈ alkyl; each Z is independentlyhydrogen or C₁-C₄ alkyl, or the two Z with the carbon atom to which theyare attached form a C═O; and in Formula I, L is selected fromsubstituted C₁-C₂₀ alkylene, C₃-C₈ cycloalkylene, substituted C₁-C₂₀alkylene wherein one or more of the —CH₂— groups are independentlyreplaced with C₃-C₈ cycloalkylene, —S—, —NR⁸C(═O)—, —SO₂NR⁸—, or—NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylene substitutedwith 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ is independentlyC₁₋₈ alkyl substituted with 1 or 2 substituents independently selectedfrom the group consisting of —SH, —S—C₁₋₄ alkyl, —CONR¹¹R¹¹, —CO₂H, and—NR¹¹R¹¹, wherein each R¹¹ is independently hydrogen, alkyl, aryl,cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic, or the two R¹¹together with the nitrogen form a heterocyclic, wherein the heterocyclicis optionally substituted with one or two oxo; in Formula I-1, L isselected from optionally substituted C₁-C₂₀ alkylene, C₃-C₈cycloalkylene, optionally substituted C₁-C₂₀ alkylene wherein one ormore of the —CH₂— groups are independently replaced with C₃-C₈cycloalkylene, —O—, —S—, —NR⁸—, —C(O)—, —C(C═O)NR⁸—, —NR⁸C(═O)—,—SO₂NR⁸—, or —NR⁸SO₂—; substituted C₁-C₂₀ alkylene is C₁-C₂₀ alkylenesubstituted with 1 to 4 —SO₃H, —P(O)(OH)₂ or R²³, wherein each R²³ isindependently C₁₋₆ alkyl optionally substituted with 1 or 2 substituentsindependently selected from the group consisting of —SH, —S—C₁₋₄ alkyl,—CONR¹¹R¹¹, —CO₂H, and —NR¹¹R¹¹, wherein each R¹¹ is independentlyhydrogen, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, andheterocyclic, or the two R¹¹ together with the nitrogen form aheterocyclic, wherein the heterocyclic is optionally substituted withone or two oxo.
 111. A method of preparing a compound of Formula Va-1:

or a pharmaceutically acceptable salt thereof, comprising contacting anantigen binding unit with a compound of Formula V-1:

or a salt thereof.
 112. The method of claim 110, wherein the antigenbinding unit is an antibody comprising SEQ ID 1 and 2, or SEQ ID 3 and4, or SEQ ID 5 and 6, or SEQ ID 7 and 8, or SEQ ID 9 and 10, SEQ ID 11and 12, or SEQ ID 13 and 14.